Cyclic di-nucleotide compounds and methods of use

ABSTRACT

Disclosed are cyclic-di-nucleotide cGAMP analogs, methods of synthesizing the compounds, pharmaceutical compositions comprising the compounds thereof, and use of compounds and compositions in medical therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/953,492, filed Apr. 15, 2018, which is a continuation ofInternational Application No. PCT/US17/023093, filed on Mar. 17, 2017,which claims the benefit of U.S. Provisional Application No. 62/310,364,filed Mar. 18, 2016, U.S. Provisional Application No. 62/355,382, filedJun. 28, 2016, and U.S. Provisional Application No. 62/396,140, filedSep. 17, 2016, the entire contents of each of which is herebyincorporated by reference herein.

TECHNICAL FIELD

The present invention provides novel cyclic di-nucleotide cGAMP analogs,pharmaceutical compositions thereof, their synthetic methods and theiruse in medical therapy. In particular, the compounds of the inventionenhance the body's immune responses by activating STING (Stimulator ofInterferon Genes) and are useful for the immunotherapy of cancer,infectious diseases and immune disorders. The compounds are also usefulas adjuvants for developing vaccines against cancer and infectiousdiseases.

BACKGROUND

Cytosolic DNA induces type-I interferons and other cytokines that areimportant for immune defense against microbial infections and malignantcells but can also result in autoimmunity. This DNA signaling pathwayrequires the adaptor protein STING (Stimulator of Interferon Genes) andthe transcription factor IRF3, but the mechanism of DNA sensing wasunclear until recently. WO 2014099824 to The University of Texasdisclosed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP(cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA.DNA transfection or DNA virus infection of mammalian cells alsotriggered cGAMP production. cGAMP bound to STING, lead to the activationof IRF3 and induction of type-I interferons including interferon-β(IFN-β). Thus, cGAMP represents the first cyclic di-nucleotide inmetazoa and it functions as an endogenous second messenger that triggersinterferon production in response to cytosolic DNA.

Through biochemical fractionation and quantitative mass spectrometry,the inventors on WO 2014099824 also identified a cGAMP synthase (cGAS),which belongs to the nucleotidyltransferase family. Overexpression ofcGAS activated the transcription factor IRF3 and induced IFN in aSTING-dependent manner. Knockdown of cGAS inhibited IRF3 activation andIFN induction by DNA transfection or DNA virus infection. cGAS bound toDNA in the cytoplasm and catalyzed cGAMP synthesis. These resultsindicate that cGAS is a cytosolic DNA sensor that induces interferons byproducing the second messenger cGAMP. The inventors on WO 2014099824also determined that the second messenger cGAMP they isolated andsynthesized contains two phosphodiester linkages, one between the 2′-OHof GMP and 5′-phosphate of AMP, and the othe between the 3′-OH of AMPand 5′-phosphate of GMP; this molecule is referred to as 2′3′-cGAMP.

Several additional patents applications in this field have henceforthpublished:

US20140205653 and US 20140341976 to Aduro Biotech disclosecyclic-di-nucleotide (CDN) compounds that activate and inhibit STING,respectively. In particular, the CDNs of the invention are provided inthe form of a composition comprising one or more cyclic purinedinucleotides which activate or inhibit STING-dependent TBK1 activationand the resulting production of type I interferon.

WO 2015077354 A1 to The University of Chicago discloses Methods andcompositions for treating cancer by intratumorally administering astimulator of interferon genes (STING) agonist. In some embodiments,there are provided compositions and methods concerning methods fortreating cancer in a subject comprising administering to the subject aneffective amount of a stimulator of interferon genes (STING) agonist,wherein the STING agonist is administered intratumorally.

WO 2015161762 to Fudan University discloses the use of cyclicdinucleotide cGAMP for preparing antitumor drugs, wherein the tumor isgastric cancer, lung cancer, colon cancer, liver cancer, prostate canceror pancreatic cancer. cGAMP was shown to inhibit the growth of humantumor cell lines in immune compromised mice.

WO 2015185565 to GlaxoSmithKline discloses a class of cyclicdinucleotide compounds, or a pharmaceutically acceptable salt andtautomers thereof, compositions, combinations and medicaments containingsaid compounds and processes for their preparation. The invention alsorelates to the use of said compounds, combinations, compositions andmedicaments, in the treatment of diseases and conditions in whichmodulation of STING is beneficial, for example inflammation, allergicand autoimmune diseases, infectious diseases, cancer and as vaccineadjuvants.

WO 2014179335 to Memorial Sloan Kettering Cancer Center disclosescompositions, methods, kits, and assays related to the use and/orexploitation of isomers of cGAMP as well as the structure of the enzymecGAS.

There is still a need for the discovery and development of new cyclicdi-nucleotide cGAMP analogs for use in medical therapy. Specifically,cGAMP analogues with better potency, stability and specificity thanendogenous cGAMP are still needed cGAMP analogues with superior safetyand efficacy in animal models of human diseases, including cancer andinfectious diseases, have yet to be developed.

SUMMARY OF THE INVENTION

Formula I encompasses Formula Ia-Ii.

In one aspect, the present invention provides a compound of Formula Ia

wherein:

a and b are independently 0 or 1 and a+b=1, when a is 1, b is 0 and R⁵is not present; and when a is 0, b is 1 and R⁴ is not present;

X¹ and X² are independently O, S or Se in a five-membered ring;

L¹, starting from the carbon alpha to X¹, and L², starting from thecarbon alpha to X², are independently —CH₂O—P(O)R⁶—O—, —CH₂O—P(S)R⁶—O—,—C(Y¹)(Y²)O—P(O)R⁶—C(Y³)(Y⁴)—, —CH₂NHSO₂NH—, —CH₂NHC(O)NH—,—CH₂NHC(S)NH—, —CH₂NHC(NH)NH—, —CH₂NHC(O)CH₂—, —CH₂NHSO₂CH₂—,—CH₂CH₂C(O)NH—, —CH₂CH₂SO₂NH—, —CH₂NH(3,4-dioxocyclobuten-1,2-diyl)NH—,

-   -   c is 0, 1, or 2;    -   d, e, and f are independently 0 or 1;    -   Y¹, Y², Y³, and Y⁴ are independently H or F;    -   R⁶ is hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), borano (—BH₃ ⁻), or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups;

R¹ and R² are independently aromatic rings or heteroaromatic rings withthe following general structure including its tautomeric forms:

-   -   g and h are independently 0 or 1;    -   W¹ and W² are independently hydrogen, halogen, hydroxyl,        C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or more        halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,        di(C₁₋₆alkyl)amino, or azido groups, C₁₋₆alkoxy, C₁₋₆alkoxy        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),        poly(ethylene glycol), azido, or —NR⁷R⁸;    -   Z¹, Z², Z³, Z⁴, Z⁵, and Z⁶ are independently CH or N;    -   if present, Z⁷, Z⁸, Z⁹, Z¹⁰, and Z¹¹ are independently CH or N,        and then W¹ is CH or N; and

R³, R⁴, and R⁵ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a compound of FormulaIb

wherein:

X¹ and X² are independently O, S or Se;

Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, and Z¹⁷ are independently CH or N;

L¹, starting from the carbon alpha to X¹, and L², starting from thecarbon alpha to X², are independently —CH₂O—P(O)R⁶—O—, —CH₂O—P(S)R⁶—O—,—C(Y¹)(Y²)O—P(O)R⁶—C(Y³)(Y⁴)—, —CH₂NHSO₂NH—, —CH₂NHC(O)NH—,—CH₂NHC(S)NH—, —CH₂NHC(NH)NH—, —CH₂NHC(O)CH₂—, —CH₂NHSO₂CH₂—,—CH₂CH₂C(O)NH—, —CH₂CH₂SO₂NH—, —CH₂NH(3,4-dioxocyclobuten-1,2-diyl)NH—,

-   -   c is 0, 1, or 2;    -   d, e, and f are independently 0 or 1;    -   Y¹, Y², Y³, and Y⁴ are independently H or F;    -   R⁶ is hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), borano (—BH₃ ⁻), or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; and

R³ and R⁴ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a compound ofFormula Ic

wherein:

Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, and Z¹⁷ are independently CH or N;

R³ and R⁴ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

-   -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, cyclic —(C₁₋₆alkyl)-, cyclic —(C₁₋₆alkyl)-        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino        groups, cyclic —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)-        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; and

R⁹ and R¹⁰ are independently hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkylselectively functionalized with one or more halogen, thiol, hydroxyl,carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups, C₁₋₆alkoxy,C₁₋₆alkoxy selectively functionalized with one or more halogen, thiol,hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups, C₃₋₅alkenyl-O—,C₃₋₅alkynyl-O—, oligo(ethylene glycol), poly(ethylene glycol), borano(—BH₃ ⁻), or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the oxygen atom in one or both of thetetrahydrofuranyl rings of Formula Ic is replaced by a sulfur or aselenium atom.

In another embodiment, the present invention provides a compound ofFormula Id

wherein:

X¹ and X² are independently O, S or Se;

L¹, starting from the carbon alpha to X¹, and L², starting from thecarbon alpha to X², are independently —CH₂O—P(O)R⁶—O—, —CH₂O—P(S)R⁶—O—,—C(Y¹)(Y²)O—P(O)R⁶—C(Y³)(Y⁴)—, —CH₂NHSO₂NH—, —CH₂NHC(O)NH—,—CH₂NHC(S)NH—, —CH₂NHC(NH)NH—, —CH₂NHC(O)CH₂—, —CH₂NHSO₂CH₂—,—CH₂CH₂C(O)NH—, —CH₂CH₂SO₂NH—, —CH₂NH(3,4-dioxocyclobuten-1,2-diyl)NH—,

-   -   c is 0, 1, or 2;    -   d, e, and f are independently 0 or 1;    -   Y¹, Y², Y³, and Y⁴ are independently H or F;    -   R⁶ is hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), borano (—BH₃ ⁻), or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups;

R³ and R⁴ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

R¹¹ and R¹² are independently selected from the group consisting of:

with at least one of R¹¹ and R¹² being

-   -   Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, Z¹⁷, Z¹⁸, Z¹⁹, Z²⁰, Z²¹, Z²², Z²³, Z²⁴,        Z²⁵, Z²⁶, Z²⁷, Z²⁸, Z²⁹, Z³⁰, Z³¹, Z³², Z³³, Z³⁴, Z³⁵, Z³⁶, and        Z³⁷ are each independently CH or N; and    -   W³, W⁴, W⁵, W⁶, W⁷, W⁸, and W⁹ are independently hydrogen,        halogen, hydroxyl, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), azido, or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a compound ofFormula Ie

wherein:

X³, X⁴, X⁵, and X⁶ are independently O, NH, CH₂, CHF, or CF₂;

R¹³ and R¹⁴ are independently selected from the group consisting of:

-   -   Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, Z¹⁷, Z¹⁸, Z¹⁹, Z²⁰, Z²¹, Z²², Z²³, Z²⁴,        Z²⁵, Z²⁶, Z²⁷, and Z²⁸ are each independently CH or N; and    -   W³, W⁴, W⁵, W⁶, and W⁷ are independently hydrogen, halogen,        hydroxyl, C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with        one or more halogen, thiol, hydroxyl, carbonyl, carboxyl,        carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,        C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups, C₁₋₆alkoxy,        C₁₋₆alkoxy selectively functionalized with one or more halogen,        thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), azido, or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups;

R³ and R⁴ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸; and

R¹⁵ and R¹⁶ are independently hydroxyl, thiol, methoxy, ethoxy, amino,N-methylamino, N,N-dimethylamino, N-ethylamino, N,N-diethylamino,N-morpholino, or borano (—BH₃ ⁻);

or a pharmaceutically acceptable salt thereof.

In another embodiment, the oxygen atom in one or both of thetetrahydrofuranyl rings of Formula Ie is replaced by a sulfur or aselenium atom.

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

[In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In another embodiment, the compound is:

In one embodiment, the present invention provides a compound of FormulaIf

wherein:

X¹ and X² are independently O, S or Se;

Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, and Z¹⁷ are independently CH or N;

L¹, starting from the carbon alpha to X¹, and L², starting from thecarbon alpha to X², are independently —CH₂O—P(O)R⁶—O—, —CH₂O—P(S)R⁶—O—,—C(Y¹)(Y²)O—P(O)R⁶—C(Y³)(Y⁴)—, —CH₂NHSO₂NH—, —CH₂NHC(O)NH—,—CH₂NHC(S)NH—, —CH₂NHC(NH)NH—, —CH₂NHC(O)CH₂—, —CH₂NHSO₂CH₂—,—CH₂CH₂C(O)NH—, —CH₂CH₂SO₂NH—, —CH₂NH(3,4-dioxocyclobuten-1,2-diyl)NH—,

-   -   c is 0, 1, or 2;    -   d, e, and f are independently 0 or 1;    -   Y¹, Y², Y³, and Y⁴ are independently H or F;    -   R⁶ is hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), borano (—BH₃ ⁻), or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; and

R³ and R⁵ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a compound of FormulaIg

wherein:

Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, and Z¹⁷ are independently CH or N;

R³ and R⁵ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

-   -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, cyclic —(C₁₋₆alkyl)-, cyclic —(C₁₋₆alkyl)-        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino        groups, cyclic —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)-        selectively functionalized with one or more halogen, thiol,        hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; and

R⁹ and R¹⁰ are independently hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkylselectively functionalized with one or more halogen, thiol, hydroxyl,carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups, C₁₋₆alkoxy,C₁₋₆alkoxy selectively functionalized with one or more halogen, thiol,hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups, C₃₋₅alkenyl-O—,C₃₋₅alkynyl-O—, oligo(ethylene glycol), poly(ethylene glycol), borano(—BH₃ ⁻), or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the oxygen atom in one or both of thetetrahydrofuranyl rings of Formula Ig is replaced by a sulfur or aselenium atom.

In another embodiment, the present invention provides a compound ofFormula Ih

wherein:

X¹ and X² are independently O, S or Se;

L¹, starting from the carbon alpha to X¹, and L², starting from thecarbon alpha to X², are independently —CH₂O—P(O)R⁶—O—, —CH₂O—P(S)R⁶—O—,—C(Y¹)(Y²)O—P(O)R⁶—C(Y³)(Y⁴)—, —CH₂NHSO₂NH—, —CH₂NHC(O)NH—,—CH₂NHC(S)NH—, —CH₂NHC(NH)NH—, —CH₂NHC(O)CH₂—, —CH₂NHSO₂CH₂—,—CH₂CH₂C(O)NH—, —CH₂CH₂SO₂NH—, —CH₂NH(3,4-dioxocyclobuten-1,2-diyl)NH—,

-   -   c is 0, 1, or 2;    -   d, e, and f are independently 0 or 1;    -   Y¹, Y², Y³, and Y⁴ are independently H or F;    -   R⁶ is hydroxyl, thiol, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), borano (—BH₃ ⁻), or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups;

R³ and R⁵ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸;

R¹ and R¹² are independently selected from the group consisting of:

with at least one of R¹¹ and R¹² being

-   -   Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, Z¹⁷, Z¹⁸, Z¹⁹, Z²⁰, Z²¹, Z²², Z²³, Z²⁴,        Z²⁵, Z²⁶, Z²⁷, Z²⁸, Z²⁹, Z³⁰, Z³¹, Z³², Z³³, Z³⁴, Z³⁵, Z³⁶, and        Z³⁷ are each independently CH or N; and    -   W³, W⁴, W⁵, W⁶, W⁷, W⁸, and W⁹ are independently hydrogen,        halogen, hydroxyl, C₁₋₆alkyl, C₁₋₆alkyl selectively        functionalized with one or more halogen, thiol, hydroxyl,        carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups,        C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one or        more halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), azido, or —NR⁷R⁸;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a compound ofFormula Ii

wherein:

X³, X⁵, X⁶, and X⁷ are independently O, NH, CH₂, CHF, or CF₂;

R¹³ and R¹⁴ are independently selected from the group consisting of:

-   -   Z¹², Z¹³, Z¹⁴, Z¹⁵, Z¹⁶, Z¹⁷, Z¹⁸, Z¹⁹, Z²⁰, Z²¹, Z²², Z²³, Z²⁴,        Z²⁵, Z²⁶, Z²⁷, and Z²⁸ are each independently CH or N; and    -   W³, W⁴, W⁵, W⁶, and W⁷ are independently hydrogen, halogen,        hydroxyl, C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with        one or more halogen, thiol, hydroxyl, carbonyl, carboxyl,        carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,        C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups, C₁₋₆alkoxy,        C₁₋₆alkoxy selectively functionalized with one or more halogen,        thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,        C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or        azido groups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene        glycol), poly(ethylene glycol), azido, or —NR⁷R⁸;        -   R⁷ and R⁸ are independently hydrogen, C₁₋₆alkyl, C₁₋₆alkyl            selectively functionalized with one or more halogen, thiol,            hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,            C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,            di(C₁₋₆alkyl)amino, or azido groups, cyclic —(C₁₋₆alkyl)-,            cyclic —(C₁₋₆alkyl)- selectively functionalized with one or            more halogen, thiol, hydroxyl, carbonyl, carboxyl,            carbonyloxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups, cyclic            —(C₁₋₆oxaalkyl)-, or cyclic —(C₁₋₆oxaalkyl)- selectively            functionalized with one or more halogen, thiol, hydroxyl,            carbonyl, carboxyl, carbonyloxyl, C₁₋₆hydroxyalkoxy, amino,            C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups;

R³ and R⁵ are independently hydrogen, halogen, hydroxyl, amino,C₁₋₆alkyl, C₁₋₆alkyl selectively functionalized with one or morehalogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₁₋₆alkoxy, C₁₋₆alkoxy selectively functionalized with one ormore halogen, thiol, hydroxyl, carbonyl, carboxyl, carbonyloxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups, C₃₋₅alkenyl-O—, C₃₋₅alkynyl-O—, oligo(ethylene glycol),poly(ethylene glycol), azido, or —NR⁷R⁸; and

R¹⁵ and R¹⁶ are independently hydroxyl, thiol, methoxy, ethoxy, amino,N-methylamino, N,N-dimethylamino, N-ethylamino, N,N-diethylamino,N-morpholino, or borano (—BH₃ ⁻);

or a pharmaceutically acceptable salt thereof.

In another embodiment, the oxygen atom in one or both of thetetrahydrofuranyl rings of Formula Ii is replaced by a sulfur or aselenium atom.

In another embodiment, the compound is:

In another embodiment, the compound is:

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients.

In another aspect, the present invention provides a method of treating adisease or condition in which modulation of STING is beneficialcomprising: administering to a patient in need thereof a compound ofFormula I or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt thereof, for use in thetreatment of a disease or condition in which modulation of STING isbeneficial.

In another aspect, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt thereof, for use in therapy.

In another aspect, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt thereof, for use in thetreatment of cancer.

In another aspect, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt thereof, or a pharmaceuticallycomposition thereof, such as a nanoparticle or a delivery vehicles thatenhances the cellular uptake, stability and efficacy of a compound ofFormula I for use in the treatment of cancer.

In another aspect, the present invention provides a method of treatingcancer comprising: administering a therapeutically effective amount of acompound of Formula I, or a pharmaceutically acceptable salt thereof, toa patient in need thereof.

In another aspect, the present invention provides the use of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, in themanufacture of a medication for the treatment of cancer.

In another aspect, the present invention provides pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, at least one further therapeutic agent, and oneor more of pharmaceutically acceptable excipients.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent foruse in therapy.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent foruse in the treatment of a disease or condition for which modulation ofSTING is beneficial.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent foruse in the treatment of cancer.

In another aspect, the present invention provides a method for treatinga disease or condition for which modulation of STING is beneficialcomprising: administering to a patient in need thereof a therapeuticallyeffective amount of a combination comprising a compound of Formula I, ora pharmaceutically acceptable salt thereof, and at least one furthertherapeutic agent.

In another aspect, the present invention provides a method of treatingcancer comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof, and at least one further therapeutic agent.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent foruse in the treatment of cancer. The therapeutic agent includes but isnot limited to immune checkpoint inhibitors, such as humanizedantibodies against PD1, PD-L1, CTLA4 and other molecules that blockeffective anti-tumor immune responses.

In another aspect, the present invention provides a method of treatingcancer comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof, and at least one further therapeutic agent. Thetherapeutic agent includes but is not limited to immune checkpointinhibitors, such as humanized antibodies against PD1, PD-L1, CTLA4 andother molecules that block effective anti-tumor immune responses.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent foruse in the treatment of cancer. The therapeutic agent includesradiation, such as high-dose radiation, which directly kills tumorcells, enhances presentation of tumor antigens and activates the STINGpathway.

In another aspect, the present invention provides a method of treatingcancer comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof, and at least one further therapeutic agent. Thetherapeutic agent includes radiation, such as high-dose radiation, whichdirectly kills tumor cells, enhances presentation of tumor antigens andactivates the STING pathway.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and at least one further therapeutic agent foruse in the treatment of cancer. The therapeutic agent includes anotherchemotherapeutic agent that selectively kills tumor cells and enhancespresentation of tumor antigens.

In another aspect, the present invention provides a method of treatingcancer comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof, and at least one further therapeutic agent. Thetherapeutic agent includes another chemotherapeutic agent thatselectively kills tumor cells and enhances presentation of tumorantigens.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, a pharmaceutical formulation including ananoparticle, and at least one further therapeutic agent for use in thetreatment of cancer. The therapeutic agent includes radiation and/oranother chemotherapeutic agent.

In another aspect, the present invention provides a method of treatingcancer comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof, a pharmaceutical formulation including a nanoparticle, andat least one further therapeutic agent for use in the treatment ofcancer. The therapeutic agent includes radiation and/or anotherchemotherapeutic agent.

In another aspect, the present invention provides a method of treatingcancer comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof, a pharmaceutical formulation including a nanoparticle, andat least one further therapeutic agent for use in the treatment ofcancer. The compound of Formula I, may be injected directly to tumors,or systemically, including injection into muscles (intramuscular), skins(subcutaneous and intra-dermal), peritoneal (intraperitoneal), lymphnodes (intralymphatic) or veins (intravenous).

In another aspect, the present invention provides a compound of FormulaI, or a pharmaceutically acceptable salt thereof, for use as a vaccineadjuvant.

In another aspect, the present invention provides a compound of FormulaI, or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, such as a nanoparticle or a delivery vehicles thatenhances the cellular uptake, stability and efficacy of a compound ofFormula I, for use as a vaccine adjuvant.

In one embodiment, the pharmaceutical composition is a vaccine.

In another embodiment, the present invention provides a method ofinducing or promoting an immune response comprising: administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising a compound of Formula I, or apharmaceutically acceptable salt thereof, as an adjuvant and a tumorantigen.

In another embodiment, the present invention provides a method ofinducing or promoting an immune response comprising: administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising a compound of Formula I, or apharmaceutical composition thereof, as an adjuvant, a tumor antigen, ora pharmaceutical composition thereof, such as a nanoparticle or adelivery vehicles that enhances the cellular uptake of the adjuvant andtumor antigen.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, as an adjuvant and an immunogen for a targetpathogen.

In another aspect, the present invention provides a compound of FormulaI, or a pharmaceutically acceptable salt thereof, for use as a vaccineadjuvant.

In another embodiment, the present invention provides a method ofinducing or promoting an immune response comprising: administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising a compound of Formula I, or apharmaceutically acceptable salt thereof, as an adjuvant and animmunogen for a target pathogen.

In another aspect, the present invention provides a vaccine adjuvantcomprising: a compound of Formula I, or a pharmaceutically acceptablesalt thereof.

In another aspect, the present invention provides an immunogeniccomposition comprising: an antigen or antigen composition and a compoundof Formula I, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides an immunogeniccomposition comprising: an antigen or antigen composition and a compoundof Formula I, or a pharmaceutically acceptable salt thereof, for use inthe treatment or prevention of a disease, including cancer andinfectious diseases.

In another aspect, the present invention provides the use of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, for themanufacture of an immunogenic composition comprising an antigen orantigen composition, for the treatment or prevention of a disease,including cancer and infectious diseases.

In another aspect, the present invention provides a method of treatingor preventing a disease comprising: administering to a patient sufferingfrom or susceptible to the disease, an immunogenic compositioncomprising an antigen or antigen composition and a compound of FormulaI, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a vaccine compositioncomprising: an antigen or antigen composition and a compound of FormulaI, or a pharmaceutically acceptable salt thereof, for use in thetreatment or prevention of a disease, including cancer and infectiousdiseases.

In another aspect, the present invention provides the use of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, for themanufacture of a vaccine composition comprising an antigen or antigencomposition for the treatment or prevention of a disease, includingcancer and infectious diseases.

In another aspect, the present invention provides a method of treatingor preventing disease comprising the administration to a patientsuffering from or susceptible to the disease, a vaccine compositioncomprising an antigen or antigen composition and a compound of FormulaI, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a compound of FormulaI, or a pharmaceutically acceptable salt thereof, for use in thetreatment of immune disorders, including autoimmune and autoinflammatorydiseases.

In another aspect, the present invention provides a compound of FormulaI, or a pharmaceutically acceptable salt thereof, or a pharmaceuticallycomposition thereof, such as a nanoparticle or a delivery vehicles thatenhances the cellular uptake, stability and efficacy of a compound ofFormula I, for use in the treatment of immune disorders, includingautoimmune and autoinflammatory diseases.

In another aspect, the present invention provides a method of treatingimmune disorders comprising: administering a therapeutically effectiveamount of a compound of Formula I, or a pharmaceutically acceptable saltthereof, to a patient in need thereof.

In another aspect, the present invention provides the use of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, in themanufacture of a medication for the treatment of immune disorders,including autoimmune and autoinflammatory diseases.

It will be appreciated that all combinations of the aboveaspects/embodiments, and other aspects/embodiments disclosed elsewhereherein, are contemplated and are further embodiments of the invention.

DETAILED DESCRIPTION

The present invention provides novel cGAMP analogs, pharmaceuticalcompositions thereof, and uses thereof in therapy. 2′3′-cGAMP is anendogenous second messenger produced by mammalian cells. It is a highaffinity ligand for STING, inducing conformational changes therein, anda potent inducer of type-I interferons. cGAS and the cGAS-cGAMP pathwayis important for triggering inflammatory responses to self and foreignDNA. As such, cGAS is important for immune defense against microbialpathogens that contain DNA and require DNA in their life cycles. Thesepathogens include DNA viruses, retroviruses including HIV, bacteriaincluding Mycobacterium tuberculosis, fungi and parasites. cGAS can alsodetect tumor DNA and is important for the body's intrinsic immunityagainst malignant cells. Activation of the cGAS-cGAMP-STING pathway isimportant for cancer immunotherapy.

As a potent inducer of type-I interferons, cGAMP (and hence the cGAMPanalogs of the present invention) provides a rational immune adjuvant.As such, a compound of Formula I or a pharmaceutically acceptable saltthereof may be used as a vaccine adjuvant, particularly with mucosalvaccines, and may be formulated with immunogens and delivered as havebeen cyclic-di-GMP and c-di-AMP as vaccine adjuvants (see, e.g.Pedersen, et al. PLoS ONE, November 2011, 6, 11, e26973; Ebensen et al.,Vaccine 29, 2011, 5210-5220; Chen et al., Vaccine 28, 2010, 3080-3085).In fact, such adjuvants are often more effective because cGAMP (and thecGAMP analogs of the present invention) is more potent than c-di-GMP ininducing interferons.

In one aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula I or a pharmaceutically acceptable saltthereof, for use in the treatment of cancer. In one embodiment, thepharmaceutical composition is a compound of Formula I. In anotherembodiment, the pharmaceutical composition is a compound of Formula I ina pharmaceutical formulation including a nanoparticle or anotherdelivery vehicle. In another embodiment, the pharmaceutical compositionis a compound of Formula I in combination with at least one furthertherapeutic agent, which includes but is not limited to immunecheckpoint inhibitors such as antibodies against PD-1, PD-L1 or CTLA-4.The therapeutic agent used in combination with a compound of Formula Ialso includes radiation of tumors or a chemotherapeutic agent thattargets tumor cells.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula I or a pharmaceutically acceptable saltthereof, as an adjuvant and an immunogen for a target pathogen. In oneembodiment, the pharmaceutical composition is a vaccine. In anotherembodiment, the present invention provides a method of inducing orpromoting an immune response comprising: administering to a patient inneed thereof an effective amount a pharmaceutical composition comprisinga compound of Formula I or a pharmaceutically acceptable salt thereof asan adjuvant and an immunogen for a target pathogen.

As used herein:

The terms “halo” and “halogen”, alone or in combination with othergroups, refers to fluoro-, chloro-, bromo- and iodo-.

The term “C₁₋₆ alkyl”, alone or in combination with other groups, refersto monovalent, linear chain or branched chain alkyl groups containingfrom 1 to 6 carbon atoms. Exemplary C₁₋₆ alkyl groups include but notlimited to methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl andtert-butyl groups. More preferred are C₁₋₄ alkyls.

The term “C₁₋₆ alkoxy” refers to, alone or in combination with othergroups, R′—O—, where R′ is C₁₋₆ alkyl.

The term “haloC₁₋₆alkyl”, alone or in combination with other groups,refers to a C₁₋₆ alkyl group substituted with one or more halosubstitutents, for example CF₃ and CH₂CF₃.

The term “a compound of the invention” or “a compound of Formula I”includes all solvates, complexes, poly morphs, radiolabeled derivatives,tautomers, stereoisomers, and optical isomers of the compounds ofFormula I and salts thereof unless otherwise specified.

The term “effective amount” means that amount of a drug orpharmaceutical agent that will elicit the biological or medical responseof a tissue, system, animal or human that is being sought, for instance,by a researcher or clinician.

The term “therapeutically effective amount” means any amount which, ascompared to a corresponding subject who has not received such amount,results in improved treatment, healing, prevention, or amelioration of adisease, disorder, or side effect, or a decrease in the rate ofadvancement of a disease or disorder. The term also includes within itsscope amounts effective to enhance normal physiological function.

The term “prophylaxis” includes prevention and refers to a measure orprocedure which is to prevent rather than cure or treat a disease.Preventing refers to a reduction in risk of acquiring or developing adisease causing at least one clinical symptom of the disease not todeveloping a subject that may be exposed to a disease-causing agent or asubject predisposed to the disease in advance of disease outset.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and dosage forms which are, within the scope ofsound medical judgment, suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation, orother problem or complication, commensurate with a reasonablebenefit/risk ratio.

The term “pharmaceutically acceptable excipients” includes all diluents,carriers, binders, glidants, and other components of pharmaceuticalformulations with which the compound of the invention is administered.

The compounds of the invention may exist in solid or liquid form. Insolid form, compound of the invention may exist in a continuum of solidstates ranging from fully amorphous to fully crystalline.

The term ‘amorphous’ refers to a state in which the material lacks longrange order at the molecular level and, depending upon the temperature,may exhibit the physical properties of a solid or a liquid. Typically,such materials do not give distinctive X-ray diffraction patterns and,while exhibiting the properties of a solid, are more formally describedas a liquid. Upon heating, a change from solid to liquid propertiesoccurs which is characterized by a change of state, typically secondorder (‘glass transition’).

The term ‘crystalline’ refers to a solid phase in which the material hasa regular ordered internal structure at the molecular level and gives adistinctive X-ray diffraction pattern with defined peaks. Such materialswhen heated sufficiently will also exhibit the properties of a liquid,but the change from solid to liquid is characterized by a phase change,typically first order (‘melting point’).

The compounds of the invention may have the ability to crystallize inmore than one form, a characteristic, which is known as polymorphism,and it is understood that such polymorphic forms (“polymorphs”) arewithin the scope of the invention. Polymorphism generally can occur as aresponse to changes in temperature or pressure or both and can alsoresult from variations in the crystallization process. Polymorphs can bedistinguished by various physical characteristics known in the art suchas x-ray diffraction patterns, solubility and melting point.

The compound of Formula I may exist in solvated and unsolvated forms. Asused herein, the term “solvate” refers to a complex of variablestoichiometry formed by a solute (in this invention, a compound ofFormula I or a salt) and a solvent. Such solvents for the purpose of theinvention may not interfere with the biological activity of the solute.The skilled artisan will appreciate that pharmaceutically acceptablesolvates may be formed for crystalline compounds wherein solventmolecules are incorporated into the crystalline lattice duringcrystallization. The incorporated solvent molecules may be watermolecules or non-aqueous such as ethanol, isopropanol DMSO, acetic acid,ethanolamine, and ethyl acetate molecules. Crystalline latticeincorporated with water molecules are typically referred to as“hydrates”. Hydrates include stoichiometric hydrates as well ascompositions containing variable amounts of water. The present inventionincludes all such solvates.

It is also noted that some compounds may form tautomers. ‘Tautomers’refer to compounds that are interchangeable forms of a particularcompound structure, and that vary in the displacement of hydrogen atomsand electrons. Thus, two structures may be in equilibrium through themovement of re electrons and an atom (usually H). For example, enols andketones are tautomers because they are rapidly interconverted bytreatment with either acid or base. It is understood that all tautomersand mixtures of tautomers of the compounds of the present invention areincluded within the scope of the compounds of the present invention.

The compounds of Formula I may be in the form of a salt. Typically, thesalts of the present invention are pharmaceutically acceptable salts.Salts encompassed within the term “pharmaceutically acceptable salts”refer to non-toxic salts of the compounds of this invention. For areview on suitable salts, see e.g., Berge et al, J. Pharm. Sci, 1977,66, 1-19. Suitable pharmaceutically acceptable salts can include acidaddition salts. A pharmaceutically acceptable acid addition salt can beformed by reaction of a compound of Formula I with a suitable inorganicor organic acid (such as hydrobromic, hydrochloric, sulfuric, nitric,phosphoric, p-toluenesulfonic, benzenesulfonic, methanesulfonic,ethanesulfonic, naphthalenesulfonic such as 2-naphthalenesulfonic),optionally in a suitable solvent such as an organic solvent, to give thesalt which is usually isolated for example by crystallization andfiltration. A pharmaceutically acceptable acid addition salt of acompound of Formula I can be, for example, a hydrobromide,hydrochloride, sulfate, nitrate, phosphate, p-toluenesulfonate,benzenesulfonate, methanesulfonate, ethanesulfonate, ornaphthalenesulfonate (e.g. 2-naphthalenesulfonate) salt. Othernon-pharmaceutically acceptable salts, e.g. trifluoroacetates, may beused, for example in the isolation of compounds of the invention, andare included within the scope of this invention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the compounds of Formula I.

While it is possible that, for use in therapy, the compound of theinvention may be administered as the raw chemical, it is possible topresent the compound of the invention as the active ingredient in apharmaceutical composition. Such compositions can be prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound. Accordingly, the invention further providespharmaceutical compositions comprising a compound of the invention andone or more pharmaceutically acceptable excipients. The excipient(s)must be acceptable in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof. In accordance with another aspect of the invention there isalso provided a process for the preparation of a pharmaceuticalcomposition including the agent, or pharmaceutically acceptable saltsthereof, with one or more pharmaceutically acceptable excipients. Thepharmaceutical composition can be for use in the treatment and/orprophylaxis of any of the conditions described herein.

Generally, the compound of the invention is administered in apharmaceutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual compound administered,the age, weight, and response of the individual patient, the severity ofthe patient's symptoms, and the like. Pharmaceutical compositions may bepresented in unit dose forms containing a predetermined amount of activeingredient per unit dose. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient, vehicle orcarrier. Typical unit dosage forms include prefilled, premeasuredampules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions.

Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered once or more than once aday. Such pharmaceutical compositions may be prepared by any of themethods well known in the pharmacy art.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, inhaled, intranasal, topical (including buccal,sublingual or transdermal), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous or intradermal) route. Suchcompositions may be prepared by any method known in the art of pharmacy,for example by bringing into association the active ingredient with thecarrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert excipient such as ethanol,glycerol, water and the like. Powders are prepared by reducing thecompound to a suitable fine size and mixing with a similarly preparedpharmaceutical excipient such as an edible carbohydrate, as, forexample, starch or mannitol. Flavoring, preservative, dispersing andcoloring agent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Excipients including glidants andlubricants such as colloidal silica, talc, magnesium stearate, calciumstearate or solid polyethylene glycol can be added to the powder mixturebefore the filling operation. A disintegrating or solubilizing agentsuch as agar-agar, calcium carbonate or sodium carbonate can also beadded to improve the availability of the medicament when the capsule isingested.

Moreover, when desired or necessary, excipients including suitablebinders, glidants, lubricants, sweetening agents, flavors,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.Tablets are formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant andpressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present invention can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, suspensions, syrups and elixirs can beprepared in dosage unit form so that a given quantity contains apredetermined amount of the compound. Syrups can be prepared bydissolving the compound in a suitably flavored aqueous solution, whileelixirs are prepared through the use of a non-toxic alcoholic vehicle.Suspensions can be formulated by dispersing the compound in a non-toxicvehicle. Solubilizers and emulsifiers such as ethoxylated isostearylalcohols and polyoxy ethylene sorbitol ethers, preservatives, flavoradditive such as peppermint oil or natural sweeteners or saccharin orother artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The composition can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of the invention may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines. Pharmaceutical compositions adapted fortransdermal administration may be presented as discrete patches intendedto remain in intimate contact with the epidermis of the recipient for aprolonged period of time.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or as enemas.

Dosage forms for nasal or inhaled administration may conveniently beformulated as aerosols, solutions, suspension drops, gels or drypowders.

Compositions for intranasal administration include aqueous compositionsadministered to the nose by drops or by pressurised pump. Suitablecompositions contain water as the diluent or carrier for this purpose.Compositions for administration to the lung or nose may contain one ormore excipients, for example one or more suspending agents, one or morepreservatives, one or more surfactants, one or more tonicity adjustingagents, one or more co-solvents, and may include components to controlthe pH of the composition, for example a buffer system. Further, thecompositions may contain other excipients such as antioxidants, forexample sodium metabisulphite, and taste-masking agents. Compositionsmay also be administered to the nose or other regions of the respiratorytract by nebulization. Intranasal compositions may permit thecompound(s) of Formula I or (a) pharmaceutically acceptable salt(s)thereof to be delivered to all areas of the nasal cavities (the targettissue) and further, may permit the compound(s) of Formula I or (a)pharmaceutically acceptable salt(s) thereof to remain in contact withthe target tissue for longer periods of time. A suitable dosing regimefor intranasal compositions would be for the patient to inhale slowlythrough the nose subsequent to the nasal cavity being cleared. Duringinhalation, the composition would be administered to one nostril whilethe other is manually compressed. This procedure would then be repeatedfor the other nostril. Typically, one or two sprays per nostril would beadministered by the above procedure one, two, or three times each day,ideally once daily. Of particular interest are intranasal compositionssuitable for once-daily administration.

The suspending agent(s), if included, will typically be present in anamount of from 0.1 to 5% (w/w), such as from 1.5% to 2.4% (w/w), basedon the total weight of the composition. Examples of pharmaceuticallyacceptable suspending agents include, but are not limited to, Avicef(microcrystalline cellulose and carboxymethylcellulose sodium),carboxymethylcellulose sodium, veegum, tragacanth, bentonite,methylcellulose, xanthan gum, carbopol and polyethylene glycols.

Compositions for administration to the lung or nose may contain one ormore excipients may be protected from microbial or fungal contaminationand growth by inclusion of one or more preservatives. Examples ofpharmaceutically acceptable anti-microbial agents or preservativesinclude, but are not limited to, quaternary ammonium compounds (forexample benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, lauralkonium chloride and myristyl picoliniumchloride), mercurial agents (for example phenylmercuric nitrate,phenylmercuric acetate and thimerosal), alcoholic agents (for examplechlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterialesters (for example esters of para-hydroxybenzoic acid), chelatingagents such as disodium edetate (EDTA) and other anti-microbial agentssuch as chlorhexidine, chlorocresol, sorbic acid and its salts (such aspotassium sorbate) and polymyxin. Examples of pharmaceuticallyacceptable anti-fungal agents or preservatives include, but are notlimited to, sodium benzoate, sorbic acid, sodium propionate,methylparaben, ethylparaben, propylparaben and butylparaben. Thepreservative(s), if included, may be present in an an amount of from0.001 to 1% (w/w), such as from 0.015% to 0.5% (w/w) based on the totalweight of the composition. Compositions (for example wherein at leastone compound is in suspension) may include one or more surfactants whichfunctions to facilitate dissolution of the medicament particles in theaqueous phase of the composition. For example, the amount of surfactantused is an amount which will not cause foaming during mixing. Examplesof pharmaceutically acceptable surfactants include fatty alcohols,esters and ethers, such as polyoxyethylene (20) sorbitan monooleate(Polysorbate 80), macrogol ethers, and poloxamers. The surfactant may bepresent in an amount of between about 0.01 to 10% (w/w), such as from0.01 to 0.75% (w/w), for example about 0.5% (w/w), based on the totalweight of the composition.

One or more tonicity-adjusting agent(s) may be included to achievetonicity with body fluids e.g. fluids of the nasal cavity, resulting inreduced levels of irritancy. Examples of pharmaceutically acceptabletonicity-adjusting agents include, but are not limited to, sodiumchloride, dextrose, xylitol, calcium chloride, glucose, glycerine andsorbitol. A tonicity-adjusting agent, if present, may be included in anamount of from 0.1 to 10% (w/w), such as from 4.5 to 5.5% (w/w), forexample about 5.0% (w/w), based on the total weight of the composition.

The compositions of the invention may be buffered by the addition ofsuitable buffering agents such as sodium citrate, citric acid,trometamol, phosphates such as disodium phosphate (for example thedodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodiumphosphate and mixtures thereof.

A buffering agent, if present, may be included in an amount of from 0.1to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of thecomposition.

Examples of taste-masking agents include sucralose, sucrose, saccharinor a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame,acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhizinate,thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, anatural flavouring agent, an artificial flavouring agent, andcombinations thereof.

One or more co-solvent(s) may be included to aid solubility of themedicament compound(s) and/or other excipients. Examples ofpharmaceutically acceptable co-solvents include, but are not limited to,propylene glycol, dipropylene glycol, ethylene glycol, glycerol,ethanol, polyethylene glycols (for example PEG300 or PEG400), andmethanol. In one embodiment, the co-solvent is propylene glycol.

Co-solvent(s), if present, may be included in an amount of from 0.05 to30% (w/w), such as from 1 to 25% (w/w), for example from 1 to 10% (w/w)based on the total weight of the composition.

Compositions for inhaled administration include aqueous, organic oraqueous/organic mixtures, dry powder or crystalline compositionsadministered to the respiratory tract by pressurised pump or inhaler,for example, reservoir dry powder inhalers, unit-dose dry powderinhalers, pre-metered multi-dose dry powder inhalers, nasal inhalers orpressurised aerosol inhalers, nebulisers or insufflators. Suitablecompositions contain water as the diluent or carrier for this purposeand may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous compositions mayalso be administered to the nose and other regions of the respiratorytract by nebulisation. Such compositions may be aqueous solutions orsuspensions or aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.

Compositions for administration topically to the nose (for example, forthe treatment of rhinitis) or to the lung, include pressurised aerosolcompositions and aqueous compositions delivered to the nasal cavities bypressurised pump. Compositions which are non-pressurised and aresuitable for administration topically to the nasal cavity are ofparticular interest. Suitable compositions contain water as the diluentor carrier for this purpose. Aqueous compositions for administration tothe lung or nose may be provided with conventional excipients such asbuffering agents, tonicity-modifying agents and the like. Aqueouscompositions may also be administered to the nose by nebulisation.

A fluid dispenser may typically be used to deliver a fluid compositionto the nasal cavities. The fluid composition may be aqueous ornon-aqueous, but typically aqueous. Such a fluid dispenser may have adispensing nozzle or dispensing orifice through which a metered dose ofthe fluid composition is dispensed upon the application of auser-applied force to a pump mechanism of the fluid dispenser. Suchfluid dispensers are generally provided with a reservoir of multiplemetered doses of the fluid composition, the doses being dispensable uponsequential pump actuations. The dispensing nozzle or orifice may beconfigured for insertion into the nostrils of the user for spraydispensing of the fluid composition into the nasal cavity.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend compositions generallycontain a powder mix for inhalation of the compound of Formula I or apharmaceutically acceptable salt thereof and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di-, orpolysaccharides (for example lactose or starch). Dry powder compositionsmay also include, in addition to the drug and carrier, a furtherexcipient (for example a ternary agent such as a sugar ester for examplecellobiose octaacetate, calcium stearate, or magnesium stearate.

Pharmaceutical compositions adapted for parental administration includeaqueous and nonaqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render thecomposition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions may include other agents conventionalin the art having regard to the type of formulation in question, forexample, those suitable for oral administration may include flavoringagents.

The compounds of Formula I and pharmaceutically acceptable salts thereofmay also be formulated with other adjuvants to modulate their activity.Such compositions may contain antibody(ies) or antibody fragment(s) oran antigenic component including but not limited to protein, DNA, liveor dead bacteria and/or viruses or virus-like particles, together withone or more components with adjuvant activity including but not limitedto aluminium salts, oil and water emulsions, heat shock proteins, lipidA preparations and derivatives, glycolipids, other TLR agonists such asCpG DNA or similar agents, cytokines such as GM-CSF or IL-12 or similaragents.

A therapeutically effective amount of the agent will depend upon anumber of factors including, for example, the age and weight of thesubject, the precise condition requiring treatment and its severity, thenature of the formulation, and the route of administration, and willultimately be at the discretion of the attendant physician orveterinarian. In particular, the subject to be treated is a mammal,particularly a human.

The agent may be administered in a daily dose. This amount may be givenin a single dose per day or more usually in a number (such as two,three, four, five or six) of sub-doses per day such that the total dailydose is the same.

Suitably, the amount of the compound of the invention administeredaccording to the present invention will be an amount selected from about0.01 mg to about 1 g per day (calculated as the free or unsaltedcompound).

The compounds of Formula I and pharmaceutically acceptable salts thereofmay be employed alone or in combination with other therapeutic agents.The compounds of Formula I and pharmaceutically acceptable salts thereofand the other pharmaceutically active agent(s) may be administeredtogether or separately and, when administered separately, administrationmay occur simultaneously or sequentially, in any order, by anyconvenient route in separate or combined pharmaceutical compositions.The amounts of the compound(s) of Formula I or pharmaceuticallyacceptable salt(s) thereof and the other pharmaceutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. Thecompound(s) of Formula I or pharmaceutically acceptable salt(s) thereofand further therapeutic agent(s) may be employed in combination byadministration simultaneously in a unitary pharmaceutical compositionincluding both compounds. Alternatively, the combination may beadministered separately in separate pharmaceutical compositions, eachincluding one of the compounds in a sequential manner wherein, forexample, the compound of the invention is administered first and theother second and vice versa. Such sequential administration may be closein time (e.g. simultaneously) or remote in time. Furthermore, it doesnot matter if the compounds are administered in the same dosage form,e.g. one compound may be administered topically and the other compoundmay be administered orally. Suitably, both compounds are administeredorally.

The combinations may be presented as a combination kit. By the term“combination kit” “or kit of parts” as used herein is meant thepharmaceutical composition or compositions that are used to administerthe combination according to the invention. When both compounds areadministered simultaneously, the combination kit can contain bothcompounds in a single pharmaceutical composition, such as a tablet, orin separate pharmaceutical compositions. When the compounds are notadministered simultaneously, the combination kit will contain eachcompound in separate pharmaceutical compositions either in a singlepackage or in separate pharmaceutical compositions in separate packages.The combination kit can also be provided by instruction, such as dosageand administration instructions. Such dosage and administrationinstructions can be of the kind that are provided to a doctor, forexample by a drug product label, or they can be of the kind that areprovided by a doctor, such as instructions to a patient.

When the combination is administered separately in a sequential mannerwherein one is administered first and the other second or vice versa,such sequential administration may be close in time or remote in time.For example, administration of the other agent several minutes toseveral dozen minutes after the administration of the first agent, andadministration of the other agent several hours to several days afterthe administration of the first agent are included, wherein the lapse oftime is not limited. For example, one agent may be administered once aday, and the other agent may be administered 2 or 3 times a day, or oneagent may be administered once a week, and the other agent may beadministered once a day and the like. It will be clear to a personskilled in the art that, where appropriate, the other therapeuticingredients(s) may be used in the form of salts, for example as alkalimetal or amine salts or as acid addition salts, or prodrugs, or asesters, for example lower alkyl esters, or as solvates, for examplehydrates, to optimize the activity and/or stability and/or physicalcharacteristics, such as solubility, of the therapeutic ingredient. Itwill be clear also that, where appropriate, the therapeutic ingredientsmay be used in optically pure form.

When combined in the same composition it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the composition and may be formulated foradministration. When formulated separately they may be provided in anyconvenient composition, conveniently, in such a manner as known for suchcompounds in the art.

When the compound of Formula I is used in combination with a secondtherapeutic agent active against the same disease, condition ordisorder, the dose of each compound may differ from that when thecompound is used alone. Appropriate doses will be readily appreciated bythose skilled in the art.

In one embodiment, the patient in the methods and uses of the presentinvention is a mammal. In another embodiment, the patient is a human.The compounds of the invention are useful in the treatment of diseasesand conditions in which modulation of STING is beneficial, includingcancer. As modulators of the immune response, the compounds of Formula Iand pharmaceutically acceptable salts thereof may also be useful, asstand-alone, in combination or as adjuvants, in the treatment ofdiseases and conditions in which modulation of STING is beneficial.

In one aspect, the disease or condition to be treated is cancer.Examples of cancer diseases and conditions in which a compound ofFormula I or pharmaceutically acceptable salt thereof, may havepotentially beneficial anti-tumor effects include cancers of the lung,bone, pancreas, skin, head, neck, uterus, ovaries, stomach, colon,breast, esophagus, small intestine, bowel, endocrine system, thyroidgland, parathyroid gland, adrenal gland, urethra, prostate, penis,testes, ureter, bladder, kidney or liver; rectal cancer; cancer of theanal region; carcinomas of the fallopian tubes, endometrium, cervix,vagina, vulva, renal pelvis, renal cell; sarcoma of soft tissue; myxoma;rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma;hepatoblastoma; angiosarcoma; hemangioma; hepatoma; fibrosarcoma;chondrosarcoma; myeloma; chronic or acute leukemia; lymphocyticlymphomas; primary CNS lymphoma; neoplasms of the CNS; spinal axistumours; squamous cell carcinomas; synovial sarcoma; malignant pleuralmesotheliomas; brain stem glioma; pituitary adenoma; bronchial adenoma;chondromatous hanlartoma; inesothelioma; Hodgkin's Disease; or acombination of one or more of the foregoing cancers.

In a further aspect, the present invention provides a compound ofFormula I or a pharmaceutically acceptable salt thereof, for use in thetreatment of cancer.

In a further aspect, the present invention provides a method of treatingcancer comprising administering to a patient in need thereof atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof.

In a further aspect, the present invention provides the use of acompound of Formula I or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for the treatment of cancer.

A compound of the invention may be employed with other therapeuticmethods of cancer treatment, e.g., in anti-neoplastic therapy,combination therapy with immune checkpoint inhibitors, otherchemotherapeutic, hormonal, antibody agents as well as surgical and/orradiation treatments.

Immune checkpoint inhibitors, such as humanized antibodies against PD-1,PD-L1 and CTLA4, have recently been shown to be highly successful intreating several types of metastatic cancer, including melanoma,non-small cell lung cancers, renal cell carcinoma and bladder cancer(Sharma and Allison, 2015, Science 348, 56). However, still only a smallpercentage of cancer patients benefit from the checkpoint inhibitortherapies, in part because insufficient number of anti-tumor immunecells, such as CD8 T cells, are generated and/or infiltrated into thetumors. Activation of the cGAS-STING pathway activates anti-tumorimmunity, including the production and infiltration of tumor-specificCD8 T cells. Therefore, cGAMP analogues are expected to functionsynergistically with immune checkpoint inhibitors and the combinationtherapies are likely to bring therapeutic benefits to a largerpercentage of cancer patients.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one immune checkpoint inhibitor.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one immune checkpoint inhibitorfor use in therapy.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or pharmaceuticallyacceptable salt thereof, and at least one immune checkpoint inhibitorfor use in treating cancer.

In a further aspect, the present invention provides the use of apharmaceutical composition comprising a compound of Formula I or apharmaceutically acceptable salt thereof, and at least one immunecheckpoint inhibitor in the manufacture of a medicament for thetreatment of cancer.

In a further aspect, the present invention provides a method of treatingcancer, comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of Formula I or a pharmaceutically acceptable saltthereof, and at least immune checkpoint inhibitor.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, at least one immune checkpoint inhibitor, andone or more of pharmaceutically acceptable carriers, diluents andexcipients.

Radiation of tumors, especially high-dose radiation such as stereotaticbody radiation therapy (SBRT), kills tumor cells with a high degree ofprecision. Dead tumor cells not only provide tumor antigens to generatetumor-specific cytotoxic T cells, but also release tumor DNA intoantigen presenting cells to activate the cGAS-STING pathway (Deng etal., 2014, Immunity 41, 843). Therefore, cGAMP analogues are expected tofunction synergistically with radiation therapies to benefit a largerpercentage of cancer patients.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, in combination with radiation therapy such asSBRT.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or pharmaceuticallyacceptable salt thereof, in combination with radiation therapy such asSBRT for use in treating cancer.

In a further aspect, the present invention provides the use of apharmaceutical composition comprising a compound of Formula I or apharmaceutically acceptable salt thereof, in combination with radiationtherapy such as SBRT in the manufacture of a medicament for thetreatment of cancer.

In a further aspect, the present invention provides a method of treatingcancer, comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of Formula I or a pharmaceutically acceptable saltthereof, in combination with radiation therapy such as SBRT.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and one or more of pharmaceutically acceptablecarriers, diluents and excipients, in combination with radiation therapysuch as SBRT for the treatment of cancer.

Anti-neoplastic agents include chemical compounds and antibodies thatkill tumor cells by inhibiting cell cycle, signal transduction, DNAmetabolism and angiogenesis and/or by promoting DNA damage, apoptosisand necrosis. These agents comprise that largest class of moleculescurrently used for cancer therapies. Anti-neoplastic agents selectivelykill tumor cells, although many of them also kill normal cells, therebygenerating severe side effects. Processing of dead tumor cell associatedantigens by antigen presenting cells leads to the generation oftumor-specific cytotoxic T cells. This process can be enhanced by cGAMPanalogues. Therefore, combination of cGAMP analogues withanti-neoplastic agents are likely to generate synergistic effects thatbenefit a larger percentage of patients.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent for usein therapy.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent for usein treating cancer.

In a further aspect, the present invention provides the use of apharmaceutical composition comprising a compound of Formula I or apharmaceutically acceptable salt thereof, and at least oneanti-neoplastic agent in the manufacture of a medicament for thetreatment of cancer.

In a further aspect, the present invention provides a method of treatingcancer, comprising: administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of Formula I or a pharmaceutically acceptable saltthereof, and at least one anti-neoplastic agent.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, at least one anti-neoplastic agent, and one ormore of pharmaceutically acceptable carriers, diluents and excipients.

Any anti-neoplastic agent that has activity versus a susceptible tumorbeing treated may be utilized in the combination. Typicalanti-neoplastic agents useful include anti-microtubule agents such asditerpenoids and vinca alkaloids; platinum coordination complexes;alkylating agents such as nitrogen mustards, oxazaphosphorines,alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such asanthracyclins, actinomycins and bleomycins; topoisomerase II inhibitorssuch as epipodophyllotoxins; antimetabolites such as purine andpyrimidine analogues and anti-folate compounds; topoisomerase Iinhibitors such as camptothecins; hormones and hormonal analogues;signal transduction pathway inhibitors; non-receptor tyrosineangiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents;and cell cycle signaling inhibitors.

Anti-microtubule or anti-mitotic agents are phase specific agents activeagainst the microtubules of tumor cells during M or the mitosis phase ofthe cell cycle. Examples of anti-microtubule agents include diterpenoidsand vinca alkaloids.

Diterpenoids, which are derived from natural sources, are phase specificanti-cancer agents that operate at the G₂/M phases of the cell cycle. Itis believed that the diterpenoids stabilize the β-tubulin subunit of themicrotubules, by binding with this protein. Disassembly of the proteinappears then to be inhibited with mitosis being arrested and cell deathfollowing. Examples of diterpenoids include paclitaxel and its analogdocetaxel.

Vinca alkaloids are phase specific anti-neoplastic agents derived fromthe periwinkle plant. Vinca alkaloids act at the M phase (mitosis) ofthe cell cycle by binding specifically to tubulin. Consequently, thebound tubulin molecule is unable to polymerize into microtubules.Mitosis is believed to be arrested in metaphase with cell deathfollowing. Examples of vinca alkaloids include vinblastine, vincristine,and vinorelbine.

Platinum coordination complexes are non-phase specific anti-canceragents, which are interactive with DNA. The platinum complexes entertumor cells, undergo, aquation and form intra- and interstrandcrosslinks with DNA causing adverse biological effects to the tumor.Examples of platinum coordination complexes include oxaliplatin,cisplatin and carboplatin.

Alkylating agents are non-phase anti-cancer specific agents and strongelectrophiles. Typically, alkylating agents form covalent linkages, byalkylation, to DNA through nucleophilic moieties of the DNA moleculesuch as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazolegroups. Such alkylation disrupts nucleic acid function leading to celldeath. Examples of alkylating agents include nitrogen mustards such ascyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such asbusulfan; nitrosoureas such as carmustine; and triazenes such asdacarbazine.

Antibiotic anti-neoplastics are non-phase specific agents, which bind orintercalate with DNA. Typically, such action results in stable DNAcomplexes or strand breakage, which disrupts ordinary function of thenucleic acids leading to cell death. Examples of antibioticanti-neoplastic agents include actinomycins such as dactinomycin,anthrocyclins such as daunorubicin and doxorubicin; and bleomycins.

Topoisomerase II inhibitors include epipodophyllotoxins.Epipodophyllotoxins are phase specific anti-neoplastic agents derivedfrom the mandrake plant. Epipodophyllotoxins typically affect cells inthe S and G₂ phases of the cell cycle by forming a ternary complex withtopoisomerase II and DNA causing DNA strand breaks. The strand breaksaccumulate and cell death follows. Examples of epipodophyllotoxinsinclude etoposide and teniposide.

Antimetabolite neoplastic agents are phase specific anti-neoplasticagents that act at S phase (DNA synthesis) of the cell cycle byinhibiting DNA synthesis or by inhibiting purine or pyrimidine basesynthesis and thereby limiting DNA synthesis. Consequently, S phase doesnot proceed and cell death follows. Examples of antimetaboliteanti-neoplastic agents include fluorouracil, methotrexate, cytarabine,mercaptopurine, thioguanine, and gemcitabine.

Camptothecins, including, camptothecin and camptothecin derivatives areavailable or under development as Topoisomerase I inhibitors.Camptothecins cytotoxic activity is believed to be related to itsTopoisomerase I inhibitory activity. Examples of camptothecins include,but are not limited to irinotecan, topotecan, and the various opticalforms of7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecindescribed below.

Hormones and hormonal analogues are useful compounds for treatingcancers in which there is a relationship between the hormone(s) andgrowth and/or lack of growth of the cancer. Examples of hormones andhormonal analogues useful in cancer treatment includeadrenocorticosteroids such as prednisone and prednisolone which areuseful in the treatment of malignant lymphoma and acute leukemia inchildren; aminoglutethimide and other aromatase inhibitors such asanastrozole, letrazole, vorazole, and exemestane useful in the treatmentof adrenocortical carcinoma and hormone dependent breast carcinomacontaining estrogen receptors; progestrins such as megestrol acetateuseful in the treatment of hormone dependent breast cancer andendometria 1 carcinoma; estrogens, and anti-estrogens such asfulvestrant, flutamide, nilutamide, bicalutamide, cyproterone acetateand 5a-reductases such as finasteride and dutasteride, useful in thetreatment of prostatic carcinoma and benign prostatic hypertrophy;anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, as well as selective estrogen receptor modulators (SERMS)useful in the treatment of hormone dependent breast carcinoma and othersusceptible cancers; and gonadotropin-releasing hormone (GnRH) andanalogues thereof which stimulate the release of leutinizing hormone(LH) and/or follicle stimulating hormone (FSH) for the treatmentprostatic carcinoma, for instance, LHRH agonists and antagonists such asgoserelin acetate and luprolide.

Signal transduction pathway inhibitors are those inhibitors, which blockor inhibit a chemical process which evokes an intracellular change. Asused herein this change is cell proliferation or differentiation. Signaltransduction inhibitors useful in the present invention includeinhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases,SH2/SH3 domain blockers, serine/threonine kinases, phosphatidylinositol-3 kinases, myoinositol signaling, and Ras oncogenes.

Several protein tyrosine kinases catalyze the phosphorylation ofspecific tyrosyl residues in various proteins involved in the regulationof cell growth. Such protein tyrosine kinases can be broadly classifiedas receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having anextracellular ligand binding domain, a transmembrane domain, and atyrosine kinase domain. Receptor tyrosine kinases are involved in theregulation of cell growth and are generally termed growth factorreceptors. Inappropriate or uncontrolled activation of many of thesekinases, i.e. aberrant kinase growth factor receptor activity, forexample by over-expression or mutation, has been shown to result inuncontrolled cell growth. Accordingly, the aberrant activity of suchkinases has been linked to malignant tissue growth. Consequently,inhibitors of such kinases could provide cancer treatment methods.Growth factor receptors include, for example, epidermal growth factorreceptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2,erbB4, ret, vascular endothelial growth factor receptor (VEGFr),tyrosine kinase with immunoglobulin-like and epidermal growth factorhomology domains (TIE-2), insulin growth factor-I (IGFI) receptor,macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblastgrowth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC),ephrin (eph) receptors, and the RET protooncogene. Several inhibitors ofgrowth receptors are under development and include ligand antagonists,antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.Growth factor receptors and agents that inhibit growth factor receptorfunction are described, e.g., in Kath, John C, Exp. Opin. Ther. Patents(2000) 10(6):803-818; Shawver et al. DDT Vol 2, No. 2 Feb. 1997; andLofts, F. J. et al in “Growth factor receptors as targets”, NewMolecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr,David, CRC press 1994, London.

Tyrosine kinases which are not growth factor receptor kinases are termednonreceptor tyrosine kinases. Non-receptor tyrosine kinases useful inthe present invention, which are targets or potential targets ofanti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focaladhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Suchnon-receptor kinases and agents which inhibit non-receptor tyrosinekinase function are described, e.g., in Sinh, S. and Corey, S. J.,(1999) Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80;and Bolen, J. B., Brugge, J. S., (1997) Annual review of Immunology. 15:371-404.

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domainbinding in a variety of enzymes or adaptor proteins including PI3-K p85subunit, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) andRas-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussedin Smithgall, T. E. (1995), Journal of Pharmacological and ToxicologicalMethods. 34(3) 125-32.

Inhibitors of Serine/Threonine Kinases include MAP kinase cascadeblockers which include blockers of Raf kinases (rafk), Mitogen orExtracellular Regulated Kinase (M EKs), and Extracellular RegulatedKinases (ERKs); and Protein kinase C family member blockers includingblockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).I kB kinase family (I KKa, I KKb), PKB family kinases, akt kinase familymembers, and TGF beta receptor kinases. Such Serine/Threonine kinasesand inhibitors thereof are described, e.g., in Yamamoto, T. et al.,(1999), Journal of Biochemistry. 126 (5) 799-803; Brodt, P et al.(2000), Biochemical Pharmacology, 60. 1101-1107; Massague, J., andWeis-Garcia, F. (1996) Cancer Surveys. 27:41-64; Philip, P. A.; andHarris, A. L. (1995), Cancer Treatment and Research. 78: 3-27; Lackey,K. et al Bioorganic and Medicinal Chemistry Letters, (10), 2000,223-226; U.S. Pat. No. 6,268,391; and Martinez-lacaci, L, et al., Int.J. Cancer (2000), 88(1), 44-52.

Inhibitors of Phosphotidyl inositol-3 Kinase family members includingblockers of PI3-kinase, ATM, DNA-PK, and Ku are also useful in thepresent invention. Such kinases are discussed, e.g., in Abraham, R. T.(1996), Current Opinion in Immunology. 8 (3) 412-8; Canman, C. E., Lim,D. S. (1998), Oncogene 17 (25) 3301-3308; Jackson, S. P. (1997),International Journal of Biochemistry and Cell Biology. 29 (7):935-8;and Zhong, H. et al. Cancer Res., (2000) 60(6), 1541-1545.

Also useful in the present invention are Myo-inositol signalinginhibitors such as phospholipase C blockers and Myoinositol analogues.Such signal inhibitors are described, e.g., in Powis, G., and KozikowskiA., (1994) New Molecular Targets for Cancer Chemotherapy ed., PaulWorkman and David Kerr, CRC press 1994, London.

Another group of signal transduction pathway inhibitors are inhibitorsof Ras Oncogene. Such inhibitors include inhibitors offarnesyltransferase, geranyl-geranyl transferase, and CAAX proteases aswell as anti-sense oligonucleotides, ribozymes and immunotherapy. Suchinhibitors have been shown to block ras activation in cells containingwild type mutant ras, thereby acting as antiproliferation agents. Rasoncogene inhibition is discussed in Scharovsky, O. G., et al. (2000),Journal of Biomedical Science. 7(4) 292-8; Ashby, M. N. (1998), CurrentOpinion in Lipidology. 9 (2) 99-102; and BioChim. Biophys. Acta, (19899)1423(3):19-30.

Antibody antagonists to receptor kinase ligand binding may also serve assignal transduction inhibitors. This group of signal transductionpathway inhibitors includes the use of humanized antibodies to theextracellular ligand binding domain of receptor tyrosine kinases.Examples include Imclone C225 EGFR specific antibody (see Green, M. C.et al, Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev.,(2000), 26(4), 269-286); Herceptin® erbB2 antibody (see Tyrosine KinaseSignalling in Breast cancer:erbB Family Receptor Tyrosine Kinases,Breast cancer Res., 2000, 2(3), 176-183); and 2CB VEGFR2 specificantibody (see Brekken, R. A. et al, Selective Inhibition of VEGFR2Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice,Cancer Res. (2000) 60, 5117-5124).

Anti-angiogenic agents such as non-receptor MEK angiogenesis inhibitorsmay also be useful, as well as those which inhibit the effects ofvascular endothelial growth factor (e.g., the anti-vascular endothelialcell growth factor antibody bevacizumab [Avastin™]), and compounds thatwork by other mechanisms (e.g., linomide, inhibitors of integrin ανβ3function, endostatin and angiostatin).

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent which isan anti-microtubule agent, platinum coordination complex, alkylatingagent, antibiotic agent, topoisomerase II inhibitor, antimetabolite,topoisomerase I inhibitor, hormones and hormonal analogue, signaltransduction pathway inhibitor, non-receptor tyrosine MEK angiogenesisinhibitor, immunotherapeutic agent, proapoptotic agent, or cell cyclesignaling inhibitor.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent selectedfrom diterpenoids and vinca alkaloids.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent which isa platinum coordination complex. In one embodiment, at least oneanti-neoplastic agent is paclitaxel, carboplatin, or vinorelbine.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent which isa signal transduction pathway inhibitor. In one embodiment, the signaltransduction pathway inhibitor is an inhibitor of a growth factorreceptor kinase VEGFR2, TIE2, PDGFR, BTK, erbB2, EGFr, IGFR-1, TrkA,TrkB, TrkC, or c-fms. In another embodiment, the signal transductionpathway inhibitor is an inhibitor of a serine/threonine kinase rafk,akt, or PKC-zeta. In another embodiment, the signal transduction pathwayinhibitor is an inhibitor of a non-receptor tyrosine kinase selectedfrom the src family of kinases. In another embodiment, the signaltransduction pathway inhibitor is an inhibitor of c-src. In anotherembodiment, the signal transduction pathway inhibitor is an inhibitor ofRas oncogene selected from inhibitors of farnesyl transferase andgeranylgeranyl transferase. In another embodiment, the signaltransduction pathway inhibitor is an inhibitor of a serine/threoninekinase selected from the group consisting of PI3K. In anotherembodiment, the signal transduction pathway inhibitor is a dualEGFr/erbB2 inhibitor, for example N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof, and at least one anti-neoplastic agent which isa cell cycle signaling inhibitor. In one embodiment, the cell cyclesignaling inhibitor is an inhibitor of CDK2, CDK4 or CDK6.

Compounds of Formula I may be prepared by methods known in the art oforganic synthesis as set forth in the schemes below and/or the specificExamples described below. In all of the methods, it is well understoodthat protecting groups for sensitive or reactive groups may be employedwhere necessary in accordance with general principles of chemistry.Protecting groups are manipulated according to standard methods oforganic synthesis (T. W. Green and P. G. M. Wuts (1999) ProtectiveGroups in Organic Synthesis, 3^(rd) edition, John Wiley & Sons). Thesegroups are removed at a convenient stage of the compound synthesis usingmethods that are readily apparent to those skilled in the art. Theselection of processes as well as the reaction conditions and order oftheir execution shall be consistent with the preparation of compounds ofFormula I.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art: Ac is acetyl; AcOH is acetic acid;Ac₂O is acetic anhydride; AIBN is 2,2′-azobisisobutyronitrile; Bn isbenzyl; BSA is N,O-bis(trimethylsilyl)acetamide; BSTFA isN,O-bis(trimethylsilyl)trifluoroacetamide; Bu is butyl; Bz is benzoyl;CAN is ceric ammonium nitrate; CE is 2-cyanoethyl; DCA is dichloroaceticacid; DCM is dichloromethane; DDTT is 1,2,4-dithiazole-5-thione; DEAD isdiethyl azodicarboxylate; DIAD is diisopropyl azodicarboxylate; DIPEA isN,N-diisopropylethylamine; DMAP is 4-(dimethylamino)pyridine; DMF isN,N-dimethylformamide; DMOCP is2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide; DMSO isdimethylsulphoxide; DMTr is 4,4′-dimethoxytrityl; EOAc is ethyl acetate;EtOH is ethanol; HMPT is hexamethylphosphorous triamide; HPLC is highperformance liquid chromatography; ibu is isobutyryl; IBX is2-iodoxybenzoic acid; Imid is imidazole; ^(i)Pr is isopropyl; KOH ispotassium hydroxide; Me is methyl; MeCN is acetonitrile; MeOH ismethanol; MTBE is methyl tert-butyl ether; Ms is methanesulfonyl; Pd/Cis palladium on activated charcoal; NIS is N-iodosuccinimide; NPE is2-(4-nitrophenyl)ethyl; PE is petroleum ether; Ph is phenyl; PMB isp-methoxybenzyl; PPh₃ is triphenylphosphine; Py is pyridine; TBAF istetra-n-butylammonium fluoride; TBAI is tetrabutylammonium iodide; TBDPSis tert-butyldiphenylsilyl; TBHP is tert-Butyl hydroperoxide; TBS istert-butyldimethylsilyl; TCDI is 1,1′-thiocarbonyldiimidazole; TDA-1 istris[2-(2-methoxyethoxy)ethyl]amine; TEA is triethylamine; Tf istrifluoromethanesulfonyl; TFA is trifluoroacetic acid; TFE is2,2,2-trifluoroethyl; THF is tetrahydrofuran; TIPS is triisopropylsilyl;TLC is thin-layer chromatography; TMS is trimethylsilyl; TMSOTf istrimethylsilyl trifluoromethanesulfonate; Tol is p-toluoyl; Tr istrityl.

Intermediate Preparations

Preparation of B1:

Step 1: Acetonide 2

To a suspension of D-Ribose (1) (160 g, 1.07 mol) in acetone (2.0 L) isadded concentrated sulfuric acid (10.7 g, 107 mmol, 5.8 mL) at 27° C.dropwise. After stirring for 12 hours, solid Sodium bicarbonate (100 g)is added. The mixture is then filtered and the filtrate is concentratedto give crude 2 (215.0 g).

Step 2: Silyl Ether 3

To a solution of crude 2 (215 g, 1.13 mol) in DCM (1.5 L) is added TBSCl(170 g, 1.13 mol) and TEA (172 g, 1.69 mol) at 0° C. After stirring at27° C. for 12 hours, the mixture is filtered, concentrated, and purifiedby silica gel column chromatography (EA/PE=1/100 to 1/50) to give 3 as acolorless oil (285 g, 83% yield).

Step 3: Deazapurine 4

To a solution of 3 (60.0 g, 197.08 mmol) and carbon tetrachloride (67.3g, 438 mmol, 42 mL) in THF (1.2 L) is added HMPT (63.0 g, 386 mmol, 70mL) dropwise at −78° C. and stirred at 27° C. for 2 hours. To anothersolution of 6-chloro-7-deazapurine (24.2 g, 158 mmol) and KOH (16.6 g,296 mmol) in MeCN (1.2 L) is added TDA-1 (6.37 g, 19.7 mmol) at 27° C.followed by the THF solution obtained above. After stirring at 27° C.for 12 hours, the reaction mixture is filtered, concentrated, andpurified by silica gel column chromatography (EA/PE=1/50 to 1/10) togive 4 as a yellow oil (15.3 g, 18% yield).

Step 4: Adenine 5

A solution of 4 (28.6 g, 64.9 mmol) in dioxane (150 mL) and ammoniumhydroxide aqueous solution (500 mL) is stirred at 120° C. for 30 hoursin a sealed autoclave. The volatiles are then removed and the aqueoussolution is extracted with EA (300 mL×3). The combined organic layersare washed with brine (100 mL), dried over anhydrous sodium sulfate,filtered, concentrated, and partially purified by silica gel columnchromatography (EA/DCM=1/1) to give a yellow foam (9.65 g). This residueis then dissolved in THF (50 mL) and treated with TBAF trihydrate (10.9g, 34.4 mmol) at 27° C. After stirring for 2 hours, the mixture isconcentrated and purified by silica gel column chromatography(EA/DCM=1/1 to 7/1) to give 5 (5.56 g, 79% yield) as yellow solid.

Step 5: Benzoate 6

To a solution of 5 (7.26 g, 23.7 mmol) in DCM (60 mL) is added Imid(4.84 g, 71.1 mmol) and TBSCl (5.36 g, 35.6 mmol) at 27° C. Afterstirring at 27° C. for 1.5 hours, water (100 mL) is added and themixture is extracted with DCM (200 mL). The organic layer is washed withbrine (100 mL), dried over anhydrous sodium sulfate, filtered,concentrated to give crude TBS-5. To a solution of the crude TBS-5obtained above in DCM (100 mL) is added benzoyl chloride (5.14 g, 36.6mmol) at 27° C. After stirring for 12 hours, water (200 mL) is added themixture is extracted with DCM (500 mL). The organic layer is dried overanhydrous sodium sulfate, filtered, concentrated, and purified by silicagel column chromatography (EA/PE=1/50 to 1/10) to give 6 as a yellowfoam (8.12 g, 64% yield).

Step 6: B1

A solution of 6 (15.2 g, 28.9 mmol) in TFA (90 mL) and DCM (20 mL) isstirred at 27° C. for 12 hours. The volatiles are then removed and theresidue is purified by silica gel column chromatography (MeOH/DCM=1/100to 1/10) to B1 as a yellow solid (10.16 g, 95% yield).

Preparation of B2:

Step 1: Aniline 8

To a solution of 5-amino-4,6-dichloropyrimidine (63.0 g, 384 mmol) inn-BuOH (300.0 mL) is added p-methoxybenzylamine (58.0 g, 423 mmol, 55mL) and DIPEA (99.3 g, 768 mmol, 134 mL). After stirring at 100-110° C.for 15 hours, the volatiles are removed before MTBE (100 mL) is added.The solid is collected by filtration and washed with EA to give 8 as anoff-white solid (55.0 g, 54% yield). (MS: [M+H]⁺ 265.0)

Step 2: Azapurine 9

To a solution of 8 (10.0 g, 37.8 mmol) in a mixture of DCM (200 mL),AcOH (100 mL), and water (100 mL) is added sodium nitrite (2.87 g, 41.6mmol, 2.3 mL) at 0° C. After stirring at 0-25° C. for 1 hour, DCM (30mL) and saturated sodium bicarbonate aqueous solution (30 mL) are added.The layers are then separated and the aqueous phase is extracted withDCM (150 mL×3). The combined organic phases are dried over anhydroussodium sulfate, filtered, concentrated, and purified by silica gelcolumn chromatography (EA/PE=1/3) to give 9 as a light yellow solid (6.0g, 88% yield). (MS: [M+H]⁺ 276.0)

Step 3: Azaadenine 10

To a solution of 9 (6.0 g, 21.8 mmol) in 1,4-dioxane (30 mL) is addedammonium hydroxide aqueous solution (30 mL). After stirring at 30-40° C.for 5 hours, the solid is collected by filtration to give 10 as a whitesolid (4.0 g, 70% yield). (MS: [M+H]⁺ 257.1)

Step 4: Azaadenine 11

To a solution of 10 (17.0 g, 66.3 mmol) in Py (100 mL) is added DMAP(8.92 g, 73.0 mmol), Imid (13.6 g, 199 mmol) and benzoyl chloride (14.0g, 99.5 mmol, 11.6 mL). After stirring at 110-120° C. for 18 hours, thevolatiles are removed and DCM (300 mL) and water (300 mL) are added. Thelayers are separated and the organic phase is dried over anhydroussodium sulfate, filtered, concentrated, and purified by silica gelcolumn chromatography (EA/PE=1:1) to give 11 as an off-white solid (17.0g, 68% yield). (MS: [M+H]⁺ 361.2)

Step 5: Azaadenine 12

To a suspension of 11 (6.40 g, 17.8 mmol) in MeCN (60 mL) is added asolution of CAN (29.2 g, 53.3 mmol) and sodium bicarbonate (1.49 g,17.76 mmol) in water (60 mL) at 0° C. After stirring at 0-25° C. for 12hours, the mixture is neutralized with sodium bicarbonate to ˜pH 7. Thesolid is collected by filtration to give 12 (2.6 g, 57% yield). (MS:[M+H]⁺ 241.1)

Step 6: Azaadenosine 14

To a solution of 12 (9.30 g, 38.7 mmol) and 13 (20.5 g, 40.7 mmol) inMeCN (350 mL) is added tin(IV) chloride (30.3 g, 116 mmol, 13.6 mL) at0° C. After stirring at 0-25° C. for 24 hours, the reaction mixture ispoured into saturated sodium bicarbonate aqueous solution (300 mL). Thesolid is filtered off and washed with water (100 mL). The filtrate isextracted with DCM (150 mL×4) and the combined organic layers are driedover anhydrous sodium sulfate, filtered, concentrated, and purified bysilica gel column chromatography (EA/DCM=1/10) to give 14 as anoff-white gum (6.10 g, 21% yield). (MS: [M+H]⁺ 684.9)

Step 7: B2

To a solution of 14 (6.1 g, 8.9 mmol) in a mixture of THF (35 mL) andMeOH (28 mL) is added lithium hydroxide aqueous solution (1M, 16.0 mL)at 0° C. After stirring at 0-25° C. for 3 hours, the mixture isneutralized with citric acid aqueous solution (1M) to ˜pH 7 and thenconcentrated and purified by silica gel column chromatography(MeOH/DCM=1/20) to give B2 as an off-white solid (2.9 g, 87% yield).(MS: [M+H]⁺ 373.1)

Preparation of DMTr-B3:

Step 1: Diol 16

To a solution of 15 (880 mg, 2.5 mmol) in Py (10 mL) is added a solutionof DMTrCl (940 mg, 2.6 mmol) in Py (5 mL). After stirring for 3 hours,the mixture is concentrated and purified by silica gel columnchromatography (MeOH/DCM=1/20 to 1/10) to provide 16 as a white foam(1.23 g, 75% yield). (MS: [M+H]⁺ 656.2)

Step 2: Alcohol 17

To a solution of 16 (900 mg, 1.4 mmol) and Imid (280 mg, 4.15 mmol) inPy (15 mL) is added TBSCl (310 mg, 2.05 mmol). After stirring for 4hours, the volatiles are removed and the residue is dissolved in DCM (50mL), washed with saturated sodium bicarbonate aqueous solution andbrine, dried over anhydrous sodium sulfate, concentrated, and purifiedby silica gel column chromatography (EA/toluene=1/3 to 2/3) to provide17 as a white solid (480 mg, 45% yield). (MS: [M+H]⁺ 770.2)

Step 3: Thiocarbamate 18

To a solution of 17 (500 mg, 0.65 mmol) in DMF (6 mL) is added TCDI (350mg, 1.94 mmol). After stirring for 2 days, EA (40 mL) and water (25 mL)are added and the layers are separated. The aqueous layer is extractedwith ethyl acetate (25 mL×3). The combined organic layers are washedwith water (20 mL), brine (20 mL×2), dried over anhydrous sodiumsulfate, and concentrated to give crude 18. (MS: [M+H]⁺ 880.2)

Step 4: Silyl Ether 19

To a degassed solution of crude 18 in toluene (10 mL) at 110° C. isadded a degassed solution of AIBN (57 mg, 0.34 mmol), tributyltinhydride (0.51 mL, 1.94 mmol) in toluene (3 mL) over 30 minutes. Afterstirring at 110° C. for 6 hours, the mixture is cooled to roomtemperature, concentrated, and purified by silica gel columnchromatography (EA/hexanes=1/5 to 2/1) to give 19 as a yellow oil (195mg, 40% yield over two steps). (MS: [M+H]⁺ 754.2)

Step 5: DMTr-B3

To a solution of 19 (190 mg, 0.252 mmol) in THF (5 mL) is added TBAF (1M in THF, 0.50 mL). After stirring at room temperature for 2 hours,water (5 mL) is added and the mixture is extracted with EA (8 mL×3),dried over anhydrous sodium sulfate, concentrated, and purified bysilica gel column chromatography (MeOH/DCM=1/20) to give DMTr-B3 as awhite solid (132 mg, 82% yield). (MS: [M+H]⁺ 640.2)

Preparation of B4:

Step 1: Alcohol 21

To a solution of 20 (12.8 g, 67.0 mmol) in Py (300 mL) is added TBDPSCl(21.0 mL, 80.4 mmol). After stirring for 3 h, MeOH (25 mL) is added andthe mixture is concentrated. The residue is dissolved in diethyl ether(200 mL), washes with sodium bicarbonate aqueous solution (10%, 100 mL)and water (100 mL), dried over anhydrous sodium sulfate, filtered,concentrated, and purified by silica gel column chromatography (diethylether/PE=1/2) to give 21 as a white solid (27.2 g, 95% yield). (MS:[M+Na]⁺ 451.2)

Step 2: Alcohol 22

A solution of 21 (27.2 g, 63.7 mmol) in DMSO (200 mL) and Ac₂O (50 mL)is stirred for 16 hour before pouring into ice water (200 mL). Themixture is extracted with diethyl ether (100 mL×3) and the combinedorganic layers are washed with sodium bicarbonate aqueous solution (10%,100 mL) and water (100 mL), and concentrated. The residue is thendissolved in MeOH (250 mL) and DCM (250 mL) at 0° C. followed byaddition of sodium borohydride (12.0 g) in 10 portions. After stirringfor 5 minutes, water (100 mL) is added and the layers are separated. Theorganic layer is then concentrated and purified by silica gel columnchromatography (diethyl ether/PE=1/2) to give 22 as a white solid (20.4g, 75% yield over two steps). (MS: [M+Na]⁺ 451.2)

Step 3: Methyl Ether 23

To a solution of 22 (4.0 g, 9.33 mmol) in DMF (45 mL) is added sodiumhydride (484 mg, 12.1 mmol) at 0° C. and stirred for 30 minutes beforemethyl iodide (0.64 mL, 10.3 mmol) is added slowly. After stirring for 3hours, water (3 mL) is added and the volatiles are removed and purifiedby silica gel column chromatography (EA/PE=1/10) to give 23 as a whitesolid (3.8 g, 92% yield). (MS: [M+Na]⁺ 465.2)

Step 4: Benzoate 24

To a solution of 23 (3.1 g, 7.0 mmol) in THF (50 mL) is added TBAF (8.4mL, 8.4 mmol) at 0° C. After stirring for 4 h at room temperature, water(5 mL) and EA are added. The layers are separated and the organic layeris washed with water and brine, concentrated, and the resulting residueis dissolved in DCM followed by addition of TEA (4.9 mL, 35 mmol) andbenzoyl chloride (0.98 mL, 8.4 mmol). After stirring for 1 hour, water(3 mL) is added and the volatiles are removed. The residue is purifiedby silica gel column chromatography (EA/PE=1/5) to give 24 as a whitesolid (1.9 g, 88% yield). (MS: [M+Na]⁺ 331.0)

Step 5: Acetate 25

A solution of 24 (0.71 g, 2.3 mmol) in HOAc (14 mL) and water (6 mL) isheated under reflux for 30 minutes. After cooling to room temperature,the mixture is co-evaporated with toluene (10 mL×4) and the resultingresidue is dissolved in Py/Ac₂O (10/1 v/v, 10 mL) followed by additionof DMAP (50 mg, 0.46 mmol). After stirring for 4 hours, the mixture isconcentrated and purified by silica gel column chromatography(EA/PE=1/3) to give 25 as a white solid (0.75 g, 92% yield). (MS:[M+Na]⁺ 375.0)

Step 6: Guanosine 27

To a suspension of 25 (500 mg, 1.42 mmol) and N²-isobutyrylguanine (500mg, 2.13 mmol) in DCM (20 mL) at 80° C. is added BSA (1.8 mL, 7.4 mmol)and stirred for 1 hour before addition of TMSOTf (0.77 mL, 4.26 mmol).After stirring at 80° C. for 3 hours, the mixture is cooled to roomtemperature before sodium bicarbonate aqueous solution (50 mL) is added.The mixture is then extracted with DCM (50 mL×3). The combined organiclayers are dried over anhydrous sodium sulfate, concentrated, andpurified by silica gel column chromatography (MeOH/DCM=1/20 to 1/10) togive 26 as a white powder (624 mg, 85% yield). (MS: [M+H]⁺ 514.2)

Step 7: B4

To a solution of 27 (0.49 g, 0.96 mmol) in MeOH/THF/water (4/5/1 v/v/v,20 mL) is added sodium hydroxide aqueous solution (10 M, 0.25 mL, 2.5mmol) at 0° C. After stirring for 30 minutes, HOAc is added and themixture is concentrated and purified by silica gel column chromatography(MeOH/DCM=1/10 to 1/5) to give B4 as an oil (322 mg, 92% yield). (MS:[M+H]⁺ 368.2)

Preparation of B5:

Step 1: Acetate 28

To a solution of 15 (7.0 g, 20 mmol) in MeCN (100 mL) is added DMAP (1.2g, 10 mmol) and Ac₂O (7.5 mL, 80 mmol) at 0° C. After stirring at roomtemperature overnight, the mixture is concentrated and purified bysilica gel flash chromatography (MeOH/DCM=1/20 to 1/10) to give 27 as awhite solid (8.77 g, 92% yield). (MS: [M+H]⁺ 480.0)

Step 2: Propargyl Ether 29

To a solution of 27 (480 mg, 1.0 mmol) in 1,4-dioxane (1 mL) is addedPPh₃ (656 mg, 2.5 mmol), propargyl bromide (0.15 mL, 2 mmol) and asolution of DEAD (0.49 mL, 2.5 mmol) in dioxane (1 mL) at 0° C. Afterstirring for 2 hours, the mixture is concentrated and purified by silicagel column chromatography (MeOH/DCM=1/50 to 1/20) to give 28 as a whitesolid (440 mg, 47% yield). (MS: [M+H]⁺ 518.2)

Step 3: B5

To a solution of 28 (150 mg, 0.17 mmol) in THF (4.5 mL) and MeOH (0.5mL) is added sodium hydroxide aqueous solution (1 M, 0.5 mL) at 0° C.After stirring for 1 hour, HOAc (0.1 mL) is added and the mixture isconcentrated and purified by silica gel column chromatography(MeOH/DCM=1/20 to 1/10) to give B5 as a white solid (40 mg, 64% yield).(MS: [M+H]⁺ 392.0)

Preparation of B6:

Step 1: Deazapurine 29

To a solution of 3 (40.0 g, 131 mmol) and carbon tetrachloride (33.6 g,218 mmol, 21 mL) in THF (500 mL) at −78° C. is added HMPT (22.5 g, 138mmol, 25 mL) over 15 min. After stirring for 2 hours with brief periodsof slight warming to prevent gel formation, the mixture is concentratedto about 70 mL. To a suspension of KOH (25.8 g, 460 mmol) in MeCN (600mL) is added TDA-1 (4.25 g, 13.14 mmol, 4.2 mL). After stirring at 25°C. for 10 minutes, 2-amino-6-chloro-7-deazapurine (22.2 g, 131 mmol) isadded. The mixture is stirred for another 10 minutes before the THFsolution obtained above is added. After stirring for 2 hours, themixture is filtered, concentrated, and purified by silica gel columnchromatography (EA/PE=3/17) to give 29 (9.20 g, 15% yield). (MS: [M+H]⁺455.3)

Step 2: Alcohol 30

To a mixture of 29 (13.7 g, 30.1 mmol) in dioxane (10 mL) is added asolution of sodium hydroxide (11.7 g, 291 mmol) in water (100 mL) at 25°C. After stirring at 80° C. for 64 hours, the mixture is cooled to 0°C., neutralized with AcOH to ˜pH 7, and extracted with EtOAc (100 mL×3).The combined organic layers are dried over anhydrous sodium sulfate,filtered, and concentrated to give crude 30. (MS: [M+1]⁺ 323.1)

Step 3: Silyl Ether 31

To a solution of crude 30 (9.7 g, 30.1 mmol) and Imid (4.1 g, 60.3 mmol)in DCM (10 mL) is added TBSCl (9.08 g, 60.3 mmol) at 25° C. Afterstirring for 16 hours, the mixture is diluted with DCM (100 mL), washedwith brine (80 mL), dried over anhydrous sodium sulfate, filtered,concentrated, and purified by silica gel column chromatography(EA/PE=1/4 to 1/1 then MeOH/DCM=1/50) to give 31 (9.0 g, 68% yield) as asolid. (MS: [M+H]⁺ 437.2)

Step 4: Isobutyrate 32

To a solution of 31 (9.0 g, 20.6 mmol) and TEA (4.2 g, 41.2 mmol) in DCM(80 mL) is added isobutyryl chloride (3.29 g, 30.9 mmol) at 0° C. Afterstirring at 25° C. for 16 hours, the mixture is diluted with DCM (100mL), washed with saturated sodium bicarbonate aqueous solution (50 mL×2)and brine (50 mL), dried over anhydrous sodium sulfate, filtered,concentrated, and purified by silica gel column chromatography(EA/PE=1/5 to 1/2) to give 32 as a white solid (4.2 g, 40% yield). (MS:[M+H]⁺ 507.2)

Step 5: B6

A solution of 32 (4.2 g, 8.29 mmol) in DCM (6 mL) and TFA (24 mL) isstirred at 25° C. for 1 hour before concentrated. The residue is thentreated with hydrogen chloride (4M in MeOH, 10 mL) at 0° C. Afterstirring at 25° C. for 10 minutes, the mixture is concentrated to givecrude B6 as a white solid (2.92 g, 99% yield). (MS: [M+H]⁺ 353.0)

Preparation of BA1:

Step 1: Proparyl Ether 34

To a solution of adenosine (33) (5.0 g, 18.7 mmol) in DMF (200 mL) at 0°C. is added sodium hydride (60% dispersion in mineral oil, 1.0 g, 25mmol) followed by the TBAI (1.5 g, 4.06 mmol) and propargyl bromide(2.12 mL, 20.9 mmol). After stirring at 55° C. for 2 days, the mixtureis purified by silica gel column chromatography (MeOH/DCM=7/93) followedby re-crystallization from ethanol to give 34 as a pale yellow solid(2.56 g, 45%).

Step 2: BA1

To a solution of 34 (1.4 g, 4.59 mmol, co-evaporated twice with Py) inPy (20 mL) is added TMSCl (2.4 mL, 18.9 mmol). After stirring for 30minutes, benzoyl chloride (0.7 mL, 6.0 mmol) is added and the mixture isstirred for 3 hours before addition of water (10 mL) and ammoniumhydroxide aqueous solution (15 mL) at 0° C. After stirring for 20minutes at room temperature, the mixture is extracted with DCM (25mL×3). The combined organic layers are dried over anhydrous sodiumsulfate, concentrated, and purified by silica gel column chromatography(MeOH/DCM=5:95) to give BA1 as a white foam (1.73 g, 92%). (MS: [M+H]⁺410.2)

Preparation of BA2:

Step 1: Pyrazolopyrimidine 36

To a solution of 35 (10.0 g, 56.5 mmol) in THF (80 mL) is added DIPEA(7.3 g, 56.5 mmol, 9.9 mL). After stirring at 0° C. for 10 minutes, asolution of hydrazine (1.81 g, 56.5 mmol, 2.0 mL) in THF (20 mL) isadded. The mixture is then stirred at 20° C. for 2 hours beforeconcentrated. After addition of DCM (100 mL) and H₂O (100 mL) to theresidue, the layers are separated and the aqueous layer is extractedwith DCM (100 mL×3). The combined organic layers are washed with brine(100 mL), dried over anhydrous sodium sulfate, filtered, concentrated,and purified by silica gel column chromatography (EA/PE=1/15 to 1/8) togive 36 as a yellow solid (3.10 g, 35% yield). (MS: [M+H]⁺ 155.1)

Step 2: Pyrazolopyrimidine 37

To a solution of 36 (200 mg, 1.29 mmol) in THF (2.0 mL) is addedammonium hydroxide (2.0 mL). After stirring at 20-30° C. for 2 hours,the mixture is concentrated, triturated with MeCN (0.5 mL), andcollected by filtration to give 37 as a red solid (100 mg, 57% yield).

Step 3: Tribenzoate 38

To a suspension of 37 (20.0 g, 148 mmol) and 13 (101 g, 200 mmol) inMeCN (1.2 L) is added boron trifluoride diethyl etherate (30.5 g, 215mmol, 26.5 mL). After stirring at 75-85° C. for 2 hours, the mixture isconcentrated and purified by silica gel column chromatography (EA/PE=1/5to 2/1) to give 38 as a yellow solid (35.0 g, 40% yield). (MS: [M+H]⁺580.3)

Step 4: Benzamide 39

To a solution of 38 (10.0 g, 17.3 mmol) in DCM (100 mL) is added DMAP(421 mg, 3.45 mmol) and TEA (5.24 g, 51.8 mmol, 7.2 mL) followed bybenzoyl chloride (2.91 g, 20.7 mmol, 2.4 mL) dropwise. After stirring at20-25° C. for 8 hours, the mixture is concentrated and purified bysilica gel column chromatography (EA/PE=1/5 to 11/) to give 39 as awhite solid (9.0 g, 76% yield). (MS: [M+H]⁺ 684.1)

Step 5: BA2

To a solution of 39 (1.0 g, 1.46 mmol) in THF (1.5 mL), MeOH (1.2 mL)and H₂O (0.3 mL) is added lithium hydroxide aqueous solution (5 M, 0.53mL). After stirring at 0-25° C. for 2 hours, the mixture is neutralizedwith citric acid (1 M) to ˜pH 7 before removal of the volatiles. Thesolid in the aqueous solution is then collected by filtration to giveBA2 as an off-white solid (300 mg, 54% yield). (MS: [M+H]⁺ 372.2)

Preparation of BA3:

Step 1: Trifluoroethyl Ether 40

To a solution of 22 (0.4 g, 0.93 mmol) in DMF (8 mL) is added sodiumhydride (48 mg, 1.12 mmol) at 0° C. and the mixture is stirred for 30minutes before 2,2,2-trifluoroethyl trifluoromethanesulfonate (0.165 mL,1.12 mmol) is added slowly. After stirring at 0° C. for 3 hours, water(3 mL) is added the mixture is concentrated and purified by silica gelcolumn chromatography (EA/hexanes=1/10) to give 40 as a white solid (218mg, 46% yield).

Step 2: Bezoate 41

To a solution of 40 (1.4 g, 2.74 mmol) in THF (25 mL) is added TBAF (3.3mL, 3.3 mmol) at 0° C. After stirring at room temperature for 4 hours,water (2 mL) is added and the mixture is extracted with EA. The organiclayer is washed with water and brine, and concentrated. The residue isthen dissolved in DCM followed by addition of and TEA (1.92 mL, 13.8mmol) benzoyl chloride (0.42 mL, 3.6 mmol). After stirring for 1 hour,water (1 mL) is added and the mixture is concentrated and purified bysilica gel column chromatography (EA/PE=1/5) to give 41 as a white solid(0.795 g, 77% yield).

Step 3: Acetate 42

A solution of 41 (0.79 g, 2.1 mmol) in HOAc (17.5 mL) and water (7.5 mL)is stirred at reflux for 30 minutes before cooled to room temperature,and co-evaporated with toluene (10 mL×4). The residue is then dissolvedin Py (12 mL) followed by addition of Ac₂O (0.8 mL, 8.4 mmol). Afterstirring for 6 hours, the mixture is concentrated and purified by silicagel column chromatography (EA/PE=1/4) to give 42 as a white solid (0.82g, 91% yield).

Step 4: Guanosine 43

To a suspension of 42 (800 mg, 1.9 mmol) and N²-isobutyrylguanine (633mg, 2.86 mmol) in dichloroethane (25 mL) at 80° C. is added BSA (2.74mL, 10.1 mmol) and stirred for 1 hour before addition of TMSOTf (1.03mL, 5.7 mmol). After stirring for 3 hours at 100° C., the mixture ispoured into sodium bicarbonate aqueous solution (60 mL) and extractedwith DCM (60 mL×3). The combined organic layers are dried over anhydroussodium sulfate, concentrated, and purified by silica gel columnchromatography (MeOH/DCM=1/20 to 1/10) to give 43 as a white solid (938mg, 85% yield).

Step 5: BA3

To a solution of 43 (0.5 g, 0.86 mmol) in MeOH (8 mL), THF (10 mL) andwater (2 mL) is added sodium hydroxide aqueous solution (10 M, 0.34 mL)at 0° C. After stirring for 30 minutes, HOAc is added and the mixture isconcentrated and purified by silica gel column chromatography(MeOH/DCM=1/10 to 1/5) to afford BA3 as an oil (348 mg, 93% yield).

Preparation of BB1

Step 1: Pyrazole 44

To a solution of 2 (75 g, 395 mmol) in MeOH (600 mL) is added hydrazinehydrate (120 mL) at 25° C. After stirring for 2 hours, the mixture isconcentrated and the residue is dissolved in EtOH (600 mL) beforeaddition of (ethoxymethylene)malononitrile (110 g, 901 mmol). Afterstirring at 78° C. for 30 minutes, the mixture is concentrated andpurified by silica gel column chromatography (EA/PE=1/20 to 1/15) togive 44 as a pale yellow solid (42 g, 38% yield).

Step 2: Thiourea 45

To a solution of 44 (16.0 g, 57 mmol) in MeOH (75 mL) and water (25 mL)is added ammonium hydroxide (280 mL) and hydrogen peroxide (150 mL).After stirring at 25° C. for 16 hours, the mixture is poured into sodiumsulfite aqueous solution (2 L) and then extracted with EA (700 mL×3).The combined organic layers are dried over anhydrous sodium sulfate,filtered, and concentrated. The residue is then dissolved in acetone (90mL) before benzoyl isothiocyanate (6.96 g, 42.7 mmol, 5.75 mL) is addedat 25° C. After stirring at 60° C. for 4 hours, the mixture isconcentrated to give crude 45 as a yellow solid.

Step 3: Pyrazolopyrimidinone 46

To a solution of the crude 45 obtained above in MeOH (150 mL) is addedsodium hydroxide aqueous solution (0.7 M, 80 mL) followed by methyliodide (6.8 g, 47.9 mmol, 3.0 mL). After stirring at 20° C. for 2 hours,the mixture is neutralized with HOAc to ˜pH 6 followed by addition ofwater (80 mL), and extracted with EA (100 mL×3). The combined organiclayers are dried over anhydrous sodium sulfate, filtered, andconcentrated. The residue is then dissolved in MeOH (30 mL) and sodiumhydroxide aqueous solution (1.4 M, 250 mL) is added. After stirring at100° C. for 2 hours, the mixture is concentrated and the residue isco-evaporated with toluene (200 mL×3) and dissolved in DCM (500 mL).Imid (18.5 g, 271 mmol), DMAP (1.66 g, 13.6 mmol), and TBSCl (40.9 g,271 mmol) are then added. After stirring at 25° C. for 18 hours,saturated sodium bicarbonate aqueous solution (1 L) is added and themixture is extracted with EA (500 mL×3). The combined organic layers aredried over anhydrous sodium sulfate, filtered, concentrated, andpurified by silica gel column chromatography (MeOH/DCM=1/60 to 1/30) togive 46 as a white solid (8.50 g, 34% yield over five steps).

Step 4: Isobutyrate 47

To a solution of 46 (23.4 g, 53.5 mmol) in Py (120 mL) is addedisobutyryl chloride (11.4 g, 107 mmol, 11.2 mL) at 25° C. After stirringat 25° C. for 16 hours, ammonium hydroxide (0.5 mL) is added and themixture is stirred for 30 minutes before concentrated. The residue isthen dissolved in EtOAc (1.5 L), washed with saturated ammoniumhydroxide aqueous solution (500 mL×3) and brine (500 mL), dried overanhydrous sodium sulfate, filtered, concentrated, and purified by silicagel column chromatography (EA/PE=1/10 to 1/3) to give 47 as a lightyellow solid (24.0 g, 88% yield). (MS: [M+Na]⁺ 530.1)

Step 5: BB1

A solution of 47 (10.0 g, 19.7 mmol) in HOAc (6 mL) and water (3 mL) isstirred at 65° C. for 5 hours. The reaction mixture is then concentratedand triturated with DCM (15 mL). The solid is collected by filtration togive crude BB1 as a white solid (4.0 g). (MS: [M+H]⁺ 354.0)

Preparation of BC1:

Step 1: Acetonide 49

To a solution of 48 (50 g, 135 mmol) in acetone (500 mL) is added2,2-dimethoxypropane (85 g, 816 mmol, 100 mL) and concentrated sulfuricacid (1.32 g, 13.5 mmol, 0.72 mL). After stirring at 25° C. for 30minute, saturated sodium bicarbonate aqueous solution (30 mL) is added.The solution is filtered, concentrated, and purified by silica gelcolumn chromatography (MeOH/DCM=1/200 to 1/50) to give 49 as a whitesolid (35 g, 63% yield). (MS: [M+H]⁺ 412.1)

Step 2: Azide 50

To a solution of 49 (5.0 g, 12.2 mmol) in Py (50 mL) is addedmethanesulfonyl chloride (2.1 g, 18 mmol, 1.4 mL) at 0° C. Afterstirring at 25° C. for 1 hour, DCM (200 mL) is added, and the solutionis washed with saturated sodium bicarbonate aqueous solution followed bybrine, dried over anhydrous sodium sulfate, and concentrated. Theresidue is then dissolved in DMF (50 mL) followed by addition of sodiumazide (3.4 g, 52.3 mmol). After stirring at 50° C. for 16 hours, DCM(400 mL) is added. The mixture is washed with water (300 mL), brine,dried over sodium sulfate, concentrate, and purified by silica gelcolumn chromatography (EA/PE=1/1) to give 50 as a light yellow solid(4.0 g). (MS: [M+H]⁺ 437.1)

Step 3: BC1

A solution of 50 (50 g, 115 mmol) in TFA (125 mL) and water (125 mL) isstirred at 25° C. for 5 hours before concentrated, co-evaporated twicewith toluene, and dissolved in MeOH (50 mL). The mixture is thenneutralized by sodium bicarbonate aqueous solution (1%) and trituratedwith MTBE. The solid is collected, washed with MTBE, dried, anddissolved in DMF (400 mL). Pd/C (10% w/w, 10 g) is then added and themixture is stirred under an atmosphere of hydrogen (15 psi) at 25° C.for 6 hours before filtered and concentrated to give crude BC1 as ayellow oil (39 g). (MS: [M+H]⁺ 371.1)

Preparation of BC2:

Step 1: Indole 52

To a solution of indole (305 mg, 2.6 mmol) in MeCN (10 mL) is addedsodium hydride (160 mg, 4.0 mmol) at 0° C. and stirred for 30 minutesbefore 51 (1.0 g, 2.6 mmol) is added. After stirring for 1 hour,saturated sodium bicarbonate aqueous solution (5 mL) is added and themixture is extracted with EA (20 mL×3). The combined organic layers arewashed with saturated sodium bicarbonate aqueous solution and brine,concentrated, and purified by silica gel column chromatography(EA/hexanes=1/4) to give 52 as a yellow oil (886 mg, 71% yield). (MS:[M+H]⁺ 470.2)

Step 2: BC2

To a solution of 52 (610 mg, 1.3 mmol) in MeOH (9 mL) is added sodiummethoxide (5.4 M in methanol, 0.54 mL). After stirring for 1 hour,hydrochloric acid (5 M, 0.5 mL) is added at 0° C. and the solution isstirred for 10 minutes before concentrated and purified by silica gelcolumn chromatography (MeOH/DCM=1/9) to give the desired product as awhite solid (197 mg, 92% yield). (MS: [M+H]⁺ 234.1)

Preparation of BC3:

Step 1: Pyrene 54

To a solution of 53 (560 mg, 2 mmol) in THF is added magnesium (54 mg,2.3 mmol) followed by a small amount of iodine. After stirring at 55° C.for 3 hours, copper(I) iodide (213 mg, 1.1 mmol) is added at 0° C. Themixture is stirred at room temperature for 45 minutes before 51 (367 mg,0.98 mmol) is added at 40° C. After stirring for 2 hours, saturatedammonium chloride aqueous solution (2 mL) and DCM (20 mL) are added. Thelayers are separated and the organic layer is washed by saturated sodiumbicarbonate aqueous solution and brine, dried anhydride sodium sulfate,concentrated, and purified by silica gel column chromatography(EA/hexanes=1/10 to 1/5) to give 54 as a white solid (52 mg, 10%).

Step 2: BC3

To a solution of 54 (230 mg, 0.4 mmol) in MeOH (5 mL) is added sodiummethoxide (30% in MeOH, 0.23 mL, 1.2 mmol) at room temperature. Afterstirring for 1 hour, saturated ammonium chloride (5 mL) is added and themixture is extracted by EA (10 mL×3). The organic layers are washed withbrine, dried over anhydrous sodium sulfate, and concentrated to givecrude BC3 as a white solid (150 mg).

Preparation of BC4:

Step 1: Alcohol 56

To a solution of 55 (91.4 g, 481 mmol) in Py (600 mL) is added tritylchloride (160.7 g, 577 mmol). After stirring at 60° C. for 16 hours, themixture is concentrated and co-evaporated with toluene for three times.The residue is partitioned between DCM (400 mL) and saturated sodiumbicarbonate aqueous solution (750 mL). The layers are separated and theaqueous phase is extracted with DCM (400 mL×2). The combined organiclayers are dried over anhydrous sodium sulfate, filtered, concentrated,and purified by silica gel column chromatography (EA/PE=1/10 to 1/5) toafford 56 as a white solid (180.5 g, 87% yield). (MS: [M+Na]⁺ 455.0)

Step 2: Ketone 57

To a solution of 56 (176 g, 407 mmol) in MeCN (1.0 L) is added IBX (228g, 814 mmol). After stirring at 90° C. for 6 hours, the mixture isfiltered and concentrated to give crude 57 as a light yellow oil (175g). (MS: [M+Na]⁺ 453.0)

Step 3: Ester 58

To a solution of sodium hydride (20.1 g, 502 mmol) in THF (1.0 L) isadded methyl 2-dimethoxyphosphorylacetate (96.3 g, 529 mmol, 76.5 mL) at0° C. dropwise over 15 minutes. After stirring for 60 minutes, crude 57(175 g) obtained above in THF (500 mL) is added dropwise at 0° C. Afterstirring at 25° C. for 16 hours, water (50 mL) is added at 0° C. and thevolatiles are removed and brine (500 mL) is added. The mixture is thenextracted with DCM (500 mL×3). The combined organic layers are driedover anhydrous sodium sulfate, filtered, and concentrated. The residue(198 g of 216 g obtained above) is then dissolved in EA (500 mL) andPd/C (10% w/w, 10 g) is added. After stirring under a hydrogenatmosphere (20 psi) at 25° C. for 16 hours, the mixture is filtered andthe filtrate is concentrated and purified by silica gel columnchromatography (EA/PE=1/15 to 1/10) to give 58 as a white solid (120 g,66% yield) (MS: [M+Na]⁺ 511.1)

Step 4: Alcohol 59

To a solution of lithium aluminum hydride (6.21 g, 164 mmol) in THF (200mL) is added 58 (20.0 g, 40.9 mmol) in THF (50 mL) slowly at 0° C. Afterstirring at 25° C. for 16 hours, the reaction is quenched by sequentialaddition of water (6.2 mL), sodium hydroxide aqueous solution (15%, 6.2mL), and water (18.6 mL). The mixture is then dried over anhydroussodium sulfate, filtered, concentrated, and purified by silica gelcolumn chromatography (EA/PE=1/10 to 1/4) to give 59 as a white solid.(MS: [M+Na]⁺ 483.2)

Step 5: Alcohol 60

To a solution of sodium hydride (60% w/w, 6.95 g, 174 mmol) in THF (200mL) is added 59 (20.0 g, 43.4 mmol) in THF (80 mL) at −20° C. dropwiseover 5 minutes. After stirring at 25° C. for 2 hours, benzyl bromide(22.3 g, 130 mmol, 15.5 mL) is added dropwise and the mixture is stirredat 80° C. for 16 hours before water (2 mL) is added at 0° C. The mixtureis diluted with water (200 mL) and extracted with DCM (200 mL×3). Thecombined organic layers are dried over anhydrous sodium sulfate,filtered and concentrated. The residue is then dissolved in DCM (200 mL)and DCA (5.48 g, 42.5 mmol, 12.0 mL) is added. After stirring at 25° C.for 3 hours, saturated sodium bicarbonate aqueous solution is added at0° C. The mixture is then extracted with DCM (150 mL×3). The combinedorganic solvent are dried over anhydrous sodium sulfate, filtered,concentrated, and purified by silica gel column chromatography(EA/PE=1/5 to 1/2) to give 60 as a yellow oil (12.1 g, 90% yield).

Step 6: Benzoate 61

To a solution of 60 (24.0 g, 78 mmol) in DCM (500 mL) is added benzoylchloride (16.4 g, 116.7 mmol, 13.6 mL) and TEA (23.6 g, 233.5 mmol, 32.4mL). After stirring at 25° C. for 16 hours, the mixture is concentratedand purified by silica gel column chromatography (EA/PE=1/15 to 1/10) togive 61 as a light yellow oil (30.0 g, 93% yield). (MS: [M+Na]⁺ 435.1)

Step 7: Acetate 62

A mixture of 61 (29.0 g, 70.3 mmol) and water (3.0 mL) in HOAc (220 mL)is stirred at 70° C. for 16 hours before saturated sodium bicarbonateaqueous solution is added. The mixture is then extracted with DCM (400mL×3). The combined organic layers are concentrated and the residue isdissolved in Py (30 mL) followed by addition of Ac₂O (28.5 g, 280 mmol,26 mL). After stirring at 20° C. for 16 hours, saturated sodiumbicarbonate aqueous solution is added and the mixture is then extractedwith DCM (500 mL×3). The combined organic layers are concentrated andpurified by silica gel column chromatography (EA/PE=1/10 to 1/5) to give62 as a white solid (31.1 g, 97% yield). (MS: [M+Na]⁺ 479.1)

Step 8: Purine 63

To a suspension of O⁶-diphenylcarbamoyl-N²-isobutyrylguanine (5.47 g,13.1 mmol) in MeCN (150 mL) is added BSA (11.6 g, 57.0 mmol, 14.1 mL) at20° C. After stirring at 63° C. for 30 minutes, the volatiles areremoved and the residue is dissolved in MeCN (200 mL) before 62 (5.00 g,11.0 mmol) in MeCN (50 mL) and TMSOTf (3.65 g, 16.4 mmol, 3.0 mL) areadded at −15° C. After stirring at 63° C. for 50 minutes, the mixture iscooled to 0° C., poured into saturated sodium bicarbonate aqueoussolution and extracted with EA (150 mL×3). The combined organic layersare dried over anhydrous sodium sulfate, and concentrated, and purifiedby silica gel flash chromatography (EA/PE=1/3 to 1/1) to give 63 as awhite solid. (MS: [M+H]⁺ 813.1)

Step 9: Guanosine 64

A solution of 63 (16.2 g, 19.9 mmol) in 90% TFA aqueous solution (60 mL)is stirred at 20° C. for 30 minutes before poured into saturated sodiumbicarbonate aqueous solution at 0° C. and extracted with EA (100 mL×4).The combined organic layers are dried over anhydrous sodium sulfate,concentrated, and purified by silica gel flash chromatography (EA/PE=1/1to 1/0) to give 64 as a white solid (11.4 g, 93% yield). (MS: [M+H]⁺618.1)

Step 10: Silyl Ether 65

To a solution of 64 (15.0 g, 24.3 mmol) in EtOH (500 mL) is added Pd/C(10% w/w, 2.0 g) and concentrated hydrochloric acid (10 drops). Afterstirring at 50° C. under an atmosphere of hydrogen (45 psi) for 15hours, the mixture is filtered and solid is washed with EtOH (100 mL×3).The filtrate is concentrated and one-third of the residue is dissolvedin DMF (60 mL) followed by addition of Imid (1.57 g, 23.0 mmol), DMAP(46.9 mg, 0.38 mmol) and triisopropylsilyl chloride (2.22 g, 11.5 mmol,2.5 mL). After stirring at 20° C. for 16 hours, saturated sodiumbicarbonate aqueous solution (20 mL) and water (100 mL) are added. Themixture is then extracted with EA (100 mL×2). The combined organiclayers are dried over anhydrous sodium sulfate, filtered, concentrated,and purified by silica gel column chromatography (EA/PE=1/5 to 2/1) togive 65 as a white solid (4.52 g, 86% yield). (MS: [M+H]⁺ 684.4)

Step 11: BC4

To a solution of 65 (3.0 g, 4.4 mmol) in EtOH (30 mL) is added sodiumhydroxide aqueous solution (2 M, 31 mL) at 0° C. After stirring at 0° C.for 30 minute, the mixture is neutralized by addition of hydrochloricacid solution (1 N) and HOAc at 0° C. Toluene (30 mL) is then added andmixture is concentrated to give crude BC4 as a white solid (3.0 g). (MS:[M+H]⁺ 538.2)

Preparation of BC5:

Step 1: Alcohol 66

To a solution of 15 (2.0 g, 5.66 mmol) in Py (56 mL) at 0° C. is added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.79 g, 5.66 mmol, 1.8mL) slowly. After stirring at 0° C. for 30 minutes and 25° C. for 12hours, the solution is concentrated and purified by silica gel columnchromatography (MeOH/DCM=1/20) to give 66 (1.6 g, 47% yield). (MS:[M+H]⁺ 596.3)

Step 2: Purine 67

To a solution of 66 (8.0 g, 13.5 mmol) and 2-(4-nitrophenyl)ethanol(3.37 g, 20.2 mmol) in THF (100 mL) is added DIAD (6.81 g, 33.7 mmol,6.6 mL) and PPh₃ (8.83 g, 33.7 mmol) at 25° C. slowly. After stirring at25° C. for 12 hours, water (5 mL) is added and the mixture isconcentrated and purified by silica gel column chromatography(MeOH/DCM=1/20) to give 67 as a pale yellow solid (4.5 g, 44% yield).(MS: [M+H]⁺ 745.3)

Step 3: Alcohol 68

To a solution of 67 (4.20 g, 5.64 mmol) in MeCN (40 mL) at 25° C. isadded IBX (3.16 g, 11.3 mmol). After stirring at 80° C. for 12 hours,the mixture is filtered and concentrated, and dissolved in THF (50 mL).Sodium triacetoxyborohydride (5.7 g, 27.0 mmol) is then added at 0° C.slowly. After stirring at 25° C. for 6 hours, water (5 mL) is added andthe mixture is concentrated and purified by silica gel columnchromatography (MeOH/DCM=1/20) to give 68 as a pale yellow oil (1.0 g,33% yield). (MS: [M+H]⁺ 745.3)

Step 4: Azide 69

To a solution of 68 (2.2 g, 2.95 mmol) and DMAP (1.44 g, 11.8 mmol) inDCM (140 mL) and Py (10 mL) is added trifluoromethanesulfonic anhydride(1.33 g, 4.72 mmol, 0.78 mL) at 0° C. slowly. After stirring at 0° C.for 1.5 hours, the mixture is concentrated. The residue is thendissolved in DMF (10 mL) and sodium azide (0.49 g, 7.53 mmol) is added.After stirring at 60° C. for 6 hours, the solution is concentrated andpurified by preparative HPLC (MeOH/water with 0.1% HCOOH: 40-100%) togive 69 as a pale yellow solid (1.50 g, 79% yield). (MS: [M+H]⁺ 770.4)

Step 5: BC5

To a solution of 69 (2.50 g, 3.25 mmol) in THF (12 mL) is added TBAF(13.1 g, 50.1 mmol) and HOAc (1.50 g, 25.0 mmol, 1.43 mL) at 15° C.slowly. After stirring at 15° C. for 12 hours, the mixture isconcentrated. The residue is then dissolved in DCM (20 mL), washed withwater (5 mL×2), dried over anhydrous sodium sulfate, filtered, andpurified by silica gel column chromatography (MeOH/DCM=1/20) to give BC5as a pale yellow solid (900 mg, 53% yield). (MS: [M+H]⁺ 528.2)

The following compounds are prepared essentially by the method forIntermediates BC3 and BC4 above.

TABLE 1 Intermediates BC6 and BC7 Refer- ence of Prepar- StartingMaterial Product ation

BC3

BC4Preparation of A1 and A2:

Step 1: Benzoate 70

To a solution of 33 (120 g, 449 mmol) in Py (1.0 L) is added TMSCl (390g, 3.59 mol, 454 mL). After stirring at 0° C. for 2 hours, benzoylchloride (316 g, 2.25 mol, 261 mL) is added dropwise and the mixture isstirred at 25° C. for 14 hours before cooled to 0° C. Water (240 mL) isthen added and the mixture is stirred at 25° C. for 30 minutes beforeammonium hydroxide (460 mL) is added at 0° C. After stirring for 2hours, the mixture is concentrated to give 70 as a white solid (150 g,90% yield).

Step 2: Diol 71

To a solution of 70 (150 g, 404 mmol) in Py (500 mL) is added DMTrCl(274 g, 808 mmol), TEA (81.8 g, 808 mmol, 112 mL) and DMAP (4.93 g, 40.4mmol) at 0° C. After stirring at 25° C. for 16 hours, saturated sodiumbicarbonate aqueous solution (1 L) is added and the mixture is extractedwith EtOAc (600 mL×3). The combined organic layers are dried overanhydrous sodium sulfate, filtered, concentrated, and purified by silicagel column chromatography (EA/PE 1/4 to 1/2 then MeOH/DCM 1/100 to 1/20)to give 71 as a white solid (65.0 g, 24% yield).

Step 3: Silyl Ether 72

To a solution of 71 (65.0 g, 96.5 mmol) in Py (500 mL) is added silvernitrate (32.8 g, 193 mmol) and TBSCl (29.1 g, 193 mmol) at 25° C. Afterstirring at 25° C. for 1 hour, saturated sodium bicarbonate aqueoussolution (1 L) is added and the mixture is extracted with EtOAc (600mL×3). The combined organic layers are dried over anhydrous sodiumsulfate, filtered, concentrated, and purified by silica gel columnchromatography (EA/PE=1/4 to 1/1) to give 72 as a white solid (20.0 g,26% yield).

Step 4: A2

To a solution of 72 (12.0 g, 15.2 mmol) in DIPEA (15 mL) and DCM (30 mL)is added DMAP (744 mg, 6.09 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (5.41 g, 22.9 mmol) at 25° C. Afterstirring for 2 hours, the mixture is purified directly by basic silicagel column chromatography (EA/PE=1/4 to 1/1) to give A2 as a white solid(13.0 g, 86% yield).

Step 5: A1

To a solution of A2 (3.8 g, 3.9 mmol) in MeCN (20 mL) is added water(0.1 mL) and pyridinium trifluoroacetate (1.1 g, 5.8 mmol) at 25° C. andstirred for 5 minutes before tert-butylamine (14.0 g, 0.19 mmol) isadded. After stirring for 15 minutes, the volatiles are removed and theresidue is dissolved in DCM (20 mL). A solution of DCA (1.9 g, 14.6mmol) in DCM (20 mL) is then added. After stirring for 30 minutes, TEA(3 mL) is added and the mixture is concentrated and purified byreverse-phase silica gel column chromatography (MeCN with 0.1%TEA/water=0% to 100%) to give A1⋅TEA salt as a white solid (1.5 g, 71%yield). (MS: [M+H]⁺ 549.2)

Preparation of AB1:

To a solution of A2 (494 mg, 0.5 mmol) and triphenylphosphine (197 mg,0.75 mmol), and 2-(tert-butyldimethylsilyloxy)ethanol (132 mg, 0.75mmol) in THF (5 mL) is added DIAD (0.15 mL, 0.75 mmol). After stirringat room temperature for 5 hours, the mixture is concentrated andpurified by silica gel column chromatography (EA/hexanes=1/9 to 1/4) togive AB1 as a white solid (230 mg, 40% yield).

Preparation of AC1:

Step 1: Diol 73

To a solution of crude BC1 (39 g) in Py (40 mL) is added DMTrCl (35.9 g,106 mmol) at 0° C. After stirring at 25° C. for 16 hours, MeOH (50 mL)is added and the mixture is concentrated. The residue is then dissolvedin DCM (600 mL), washed with saturated sodium bicarbonate aqueoussolution and brine, concentrated, and purified by silica gel columnchromatography (MeOH/DCM=1/100 to 1/50) to give 73 as a pale yellowsolid (34.0 g, 48% yield over two steps). (MS: [M+H]⁺ 673.2)

Step 2: Silyl Ether 74

To a solution of 73 (1.0 g, 1.49 mmol) in Py (10 mL) is added silvernitrate (380 mg, 2.24 mmol, 0.38 mL) at 0° C. After stirring for 15minutes, TBSCl (270 mg, 1.79 mmol) is added and the mixture is stirredat 25° C. for 2 hours before saturated sodium bicarbonate aqueoussolution is added. The mixture is then extracted with EA (10 mL) and theorganic layer is washed with brine, concentrated, and purified by silicagel column chromatography (EA/PE=1/5) to give 74 as a pale yellow solid(400 mg, 34% yield). (MS: [M+H]⁺ 787.3)

Step 3: AC1

To a solution of 74 (1.0 g, 1.27 mmol) in Py (10 mL) is added diphenylphosphite (80%, 744 mg, 2.54 mmol, 0.61 mL). After stirring at 20° C.for 1 hour, EA (2 mL) and saturated sodium bicarbonate aqueous solution(2 mL) are added and the mixture is stirred for 1 hour. The layers areseparated and the organic layer is concentrated. The residue is thendissolved in DCM (1.0 mL) and DCA (164 mg, 1.27 mmol, 0.1 mL) is added.After stirring at 25° C. for 30 minutes, TEA (1 mL) is added and thesolution is concentrated and purified by reverse-phase silica gel columnchromatography (MeCN with 0.1% TEA/water=0% to 100%) to give AC1 as awhite solid (500 mg, 72% yield). (MS: [M+H]⁺ 549.2)

Preparation of AC2

Step 1: Alcohol 75

To a solution of BC2 (348 mg, 1.49 mmol) in Py (15 mL) is added DMAP (18mg, 0.15 mmol) and DMTrCl (0.66 g, 1.94 mmol). After stirring overnight,MeOH (3 mL) is added and the mixture is concentrated and purified bysilica gel column chromatography (EA/hexanes=1/4) to give 75 (662 mg,83% yield). (MS: [M+H]⁺ 536.2)

Step 2: AC2

To a solution of 75 (0.2 g, 0.37 mmol) in DCM (4 mL) is added DIEPA(0.15 g, 1.2 mmol, 0.2 mL) and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (0.14 g, 0.56 mmol, 0.13 mL). Afterstirring for 4 hours, the mixture is concentrated and purified by silicagel column chromatography (EA/hexanes with 1% TEA=1/4) to give AC2 (232mg, 85% yield). (MS: [M-N^(i)Pr₂+H₂O]⁺ 653.2)

Preparation of G1 and G2:

Step 1: Silyl Ether 76

To a solution of 16 (11.0 g, 16.8 mmol) in DCM (80 mL) is added TBSCl(7.59 g, 50.3 mmol) and Imid (3.43 g, 50.3 mmol). After stirring at 25°C. for 16 hours, sodium bicarbonate aqueous solution (5%, 30 mL) isadded and the mixture is extracted with DCM (60 mL×3). The combinedorganic layers are washed with brine (100 mL), dried over anhydroussodium sulfate, filtered, concentrated, and purified by basic silica gelcolumn chromatography (EA/PE=1/5 to 1/1) to 76 as a white solid (2.1 g,16% yield).

Step 2: G2

To a solution of 76 (900 mg, 1.17 mmol) in THF (4.0 mL) and DIEPA (4.0mL) is added DMAP (14.3 mg, 0.12 mmol) followed by 2-cyanoethylN,N-diisopropylchlorophosphoramidite (415 mg, 1.76 mmol) dropwise at 0°C. After stirring at 20-25° C. for 2 hours, sodium bicarbonate aqueoussolution (5%, 15 mL) is added at 0° C. The mixture is then diluted withwater (15 mL) and extracted with EA (15 mL×3). The combined organiclayers are washed with brine (10 mL), dried over anhydrous sodiumsulfate, filtered, concentrated, and purified by silica gel columnchromatography (acetone/PE=1/10 to 1/3) to give G2 as a white solid (600mg, 53% yield).

Step 3: G1

To a solution of G1 (7.0 g, 7.22 mmol) in MeCN (30 mL) is added water(0.11 mL) and pyridinium trifluoroacetate (4.18 g, 21.7 mmol) at 25° C.After stirring at 25° C. for 15 minutes, tert-butylamine (37 mL) isadded and the mixture is stirred at 25° C. for 45 minutes beforeconcentrated. The residue is then dissolved in DCM (30 mL) and asolution of DCA in DCM (6% v/v, 30 mL) is added dropwise. After stirringat 20-25° C. for 30 minutes, DCM (30 mL) and TEA (4 mL) are added. Themixture is then concentrated, dissolved in a mixture of MeCN (5 mL) andwater (5 mL), and purified by C18 reverse-phase medium pressure liquidchromatography (MeCN with 0.1% TEA/water=0% to 60%) to give G1⋅TEA saltas a yellow solid (2.30 g, 56% yield). (MS: [M+H]⁺ 532.3)

Preparation of GB1 and GB2:

Step 1: Diol 77

To a solution of two batches of the crude BB1 (8.0 g) obtained above inPy (50 mL) is added DMTrCl (9.2 g, 27.2 mmol). After stirring at 20-30°C. for 1 hour, MeOH (10 mL) is added and the mixture is concentrated andpurified by silica gel column chromatography (EA/PE=1/5 toMeOH/DCM=1/20) to give 77 as a yellow solid (11.0 g, 30% yield over twosteps). (MS: [M+Na]⁺ 678.2)

Step 2: Silyl Ether 78

To a solution of 77 (9.0 g, 14.0 mmol) in Py (50 mL) is added TBSCl(2.48 g, 16.5 mmol) and silver nitrate (5.83 g, 34.3 mmol). Afterstirring at 25-30° C. for 30 minutes, saturated sodium bicarbonateaqueous solution is added. The mixture is then extracted with DCM (200mL×2), and the combined organic layers are washed with water (50 mL),brine (50 mL), dried over anhydrous sodium sulfate, filtered,concentrated, and purified by silica gel column chromatography(EA/PE=1/10 to 1/5 to 1/3) to give 78 as a white foam (1.50 g, 14%yield). (MS: [M+Na]⁺ 792.2)

Step 3: GB1

To a solution of 78 (2.50 g, 3.3 mmol) in DIEPA (5 mL) and DCM (5 mL) isadded DMAP (200 mg, 1.62 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (1.0 g, 4.22 mmol). After stirringat 20-25° C. for 2 hours, the mixture is concentrated and purified bysilica gel column chromatography (EA/PE=1/10 to 1/4) to give GB1 as awhite foam (2.30 g, 73% yield).

Step 4: GB2

To a solution of GB1 (2.30 g, 2.4 mmol) in MeCN (2.0 mL) is added water(0.11 mL, 6.1 mmol) and pyridinium trifluoroacetate (687 mg, 3.56 mmol)at 25° C. After stirring at 25-30° C. for 30 minutes, the mixture isconcentrated and the residue is dissolved in MeCN (20 mL) beforetert-butylamine (10.5 g, 144 mmol, 15.0 mL) is added. After stirring at25-30° C. for 30 minutes, the mixture is concentrated and DCM (20 mL)followed by addition of a solution of DCA in DCM (6% v/v, 18.2 mL). Themixture is stirred at 25-30° C. for 30 minutes before neutralized by TEAto ˜pH 7, concentrated, and purified by C18 reverse-phase silica gelcolumn chromatography (MeCN with 0.1% TEA/water=0% to 40%) to give GB2as a white solid (800 mg, 63% yield). (MS: [M+H]⁺ 532.0)

Preparation of GC1

Step 1: Alcohol 79

To a solution of BC4 (1.97 g, 2.4 mmol) in Py (20 mL) is added DMTrCl(984 mg, 2.90 mmol) at 25° C. After stirring for 3 hours, MeOH (30 mL)is added and the mixture is concentrated, and purified by basic silicagel flash chromatography (EA/PE=1/5 to 4/1) to give 79 as a light yellowpowder (1.65 g, 82% yield). (MS: [M+H]⁺ 840.2)

Step 2: GC1

To a solution of 79 (2.0 g, 2.38 mmol) in Py (15 mL) is added diphenylphosphite (1.7 g, 7.1 mmol, 1.4 mL). After stirring at 20° C. for 30minutes, saturated sodium bicarbonate aqueous solution (30 mL) is addedand the mixture is stirred for 1 hour. The mixture is then extractedwith EA (30 mL×3). The combined organic layers are washed with saturatedsodium bicarbonate aqueous solution (20 mL) and brine (20 mL), driedover anhydrous sodium sulfate, and concentrated. The residue is thendissolved in DCM (40 mL) followed by addition of water (0.4 mL) and DCA(6% v/v in DCM, 40 mL). After stirring at 20° C. for 15 minutes, themixture is neutralized to ˜pH 7 by TEA, concentrated, and purified byreverse-phase silica gel column chromatography (MeCN/water=25% to 90%)to give GC1 as a white solid (1.3 g, 90% yield). (MS: [M+H]⁺ 602.1)

Preparation of GC2:

Step 1: Alcohol 80

To a solution of BC5 (900 mg, 1.71 mmol) in Py (10 mL) is added DMTrCl(809 mg, 2.39 mmol) at 15° C. After stirring at 15° C. for 12 hours,MeOH (0.5 mL) is added and the mixture is concentrated and purified bysilica gel column chromatography (MeOH/DCM=1/50) to give 80 as a yellowoil (1.0 g, 71% yield). (MS: [M+H]⁺ 528.2)

Step 2: GC2

To a solution of 80 (2.05 g, 2.47 mmol) in DMF (5.0 mL) is added Imid(673 mg, 9.88 mmol) and TBSCl (745 mg, 4.94 mmol, 0.61 mL) at 15° C.After stirring at 15° C. for 12 hours, the mixture is concentrated andthe residue is triturated with water (10 mL). The solid is thencollected and washed with water (10 mL×2), PE (10 mL×2), dried, anddissolved in THF (18 mL) before PPh₃ (1.11 g, 4.24 mmol) is added at 15°C. After stirring at 15° C. for 2.5 hours, water (0.16 mL) is added andthe mixture is stirred at 50° C. for 12 hours. The solution is thenconcentrated and purified by reverse-phase preparative-HPLC (MeOH with0.1% TEA/water=20% to 80%), to give GC2 as a white solid (900 mg, 58%yield). (MS: [M+H]⁺ 918.1)

The following compounds are prepared essentially by the method forIntermediates A1, A2, AC1, AC2, G1, G2, GC1, and GC2 above.

TABLE 2 Intermediates A1 to A5, AA1, AA2, AB1, AC1 to AC6, G1 to G7,GA1, GB1 to GB3, and GC1 to GC5 Reference of Starting Material ProductPreparation

A1 33

A2

A1 B1

A2

A2

A2

A2 A2

AB1

AC1

AC2 B1

G1 33

G1

AC2

AC2

G1 37

G2

G1

G1

G2 DMTr-B3

G2

G2

G2

GB1

GB2

GB2

GC1

GC2

G1

A2

GC1Preparation of C1

To a solution of crude G1 (obtained from 187 mg of G2⋅TEA salt, 0.2mmol, containing Py⋅DCA salt) in MeCN (0.5 mL) is added a solution of A2(0.26 g, 0.26 mmol) in MeCN (0.2 mL). After stirring for 30 minutes,DDTT (46 mg, 0.22 mmol) is added and the mixture is stirred for 1 hourbefore concentrated. The residue is dissolved in DCM (4.8 mL) and water(0.036 mL) and DCA (6% in DCM, 4.8 mL) are added. After stirring for 10minutes, Py (1 mL) is added and the mixture is concentrated and purifiedby silica gel column chromatography (MeOH/DCM=1/10 to 1/5) to give C1⋅Pysalt as a white solid (86 mg, 35% yield). (MS: [M+H]⁺ 1148.0)

Preparation of C2

To a mixture of A1 (1.0 g, 1.82 mmol, co-evaporated MeCN 20 mL×3) and G2(2.3 g, 2.37 mmol, co-evaporated with MeCN 20 mL×3) is added tetrazole(0.45 M in MeCN, 10 mL) at 25° C. and stirred for 1 hour beforeelemental sulfur (1.75 g, 6.84 mmol) is added. After stirring for 1hour, MeCN (20 mL) is added and the mixture is filtered andconcentrated. The residue is dissolved in DCM (100 mL) and DCA (1.96 g,15.2 mmol, 1.25 mL) is added. After stirring at 25° C. for 2 hours,saturated sodium bicarbonate aqueous solution (100 mL) is added. Thelayers are separated and the aqueous layer is extracted with EA (100mL×3). The combined organic layers are dried over anhydrous sodiumsulfate, filtered, concentrated, and purified by reverse-phase silicagel column chromatography (MeCN with 0.1% TEA/water=0% to 100%) to giveC2⋅TEA salt as a white solid (100 mg, 5% yield). (MS: {[M+2H]²⁺}/2574.6)

Preparation of C3:

To a solution of A2 (510 mg, 0.52 mmol, co-evaporated with MeCN 5 mL×3)in MeCN (1 mL) treated with 3 Å MS (100 mg) for 30 minutes is added amixture of G1 (250 mg, 0.47 mmol, co-evaporated with MeCN 5 mL×3) andpyridinium trifluoroacetate (109 mg, 0.56 mmol, co-evaporated with MeCN5 mL×3) in MeCN (1.5 mL) treated with 3 Å MS (50 mg) for 30 minutes.After stirring for 4 hours, TBHP (5.5 M in decane, 0.26 mL) is added andthe mixture is stirred for 30 minutes before sodium bisulfite aqueoussolution (33%, 0.24 mL) is added at 0° C. The mixture is then stirred atroom temperature for 10 minutes before filtered and concentrated. Theresidue is dissolved in DCM (6.2 mL) followed by addition of water (0.09mL) and DCA (0.37 mL) in DCM (6.2 mL). After stirring at roomtemperature for 10 minutes, Py (0.73 mL, 9.05 mmol) and DCM (35 mL) areadded. The mixture is washed with water (10 mL×2) and the combinedaqueous layers are extracted by dichloromethane (10 mL×2). The combinedorganic layers are dried over anhydrous sodium sulfate, concentrated,and purified by silica gel column chromatography(MeOH/CH₂Cl₂/Py=10:89.5:0.5 to 25:74.5:0.5) to give C3 as a white solid(250 mg, 47%). (MS: [M+H]⁺ 1132.2)

Preparation of CA1:

To a solution of G1 (500 mg, 0.94 mmol, co-evaporated with MeCN 10 mL×3)and pyridinium trifluoroacetate (218 mg, 1.13 mmol, co-evaporation withMeCN 10 mL×3) in MeCN (3 mL) treated with 3 Å MS (100 mg) for 30 minutesis added a solution of A2 (976 mg, 0.99 mmol, co-evaporated with MeCN 10mL×3) in MeCN (2 mL) treated with 3 Å MS (200 mg) for 30 minutes. Afterstirring at room temperature for 2.5 hours, the mixture is concentratedand co-evaporated with MeCN (10 mL×2). The residue is then dissolved inDCM (20 mL) followed by addition of borane dimethyl sulfide complex (2 Min THF, 0.94 mL, 1.88 mmol) dropwise. After stirring at room temperaturefor 1 hour, MeOH (0.17 mL) is added at 0° C. and stirred for 20 minutesbefore concentrated to give crude CA1.

Preparation of CC1

Step 1: Squaramide 81

To a solution of GC2 (802 mg, 0.87 mmol) in DMF (5 mL) is added3,4-dimethoxy-3-cyclobutene-1,2-dione (186 mg, 1.31 mmol) at 15° C.slowly. After stirring at 15° C. for 2 hours, the mixture isconcentrated and purified by reverse-phase preparative HPLC (MeCN with0.1% TEA/water=0% to 100%) to give a mixture of the desired product andan unidentified byproduct (0.7 g, ca. 84% purity). (MS: [M+H]⁺ 1028.4)

Step 2: CC1

To a solution of 81 (ca. 84% pure, 0.6 g) obtained above and AC1 (0.64g, 1.17 mmol) in DMF (5.0 mL) is added TEA (177 mg, 1.75 mmol, 0.24 mL)at 15° C. After stirring at 15° C. for 12 hours, the mixture isconcentrated and the residue is dissolved in DCM (5.0 mL) before DCA(470 mg, 3.65 mmol, 0.3 mL) is added. The mixture is then stirred at 15°C. for 15 minutes before concentrated. The residue is purified byreverse-phase preparative HPLC (MeCN with 0.1% TEA/water=0% to 100%) togive CC1 as a while solid (0.54 g, 40% yield over two steps). (MS:[M+H]⁺ 1242.3)

Preparation of CC2

Step 1: Sulfamate 82

To a solution of 4-nitrophenyl chlorosulfate (0.31 g, 1.31 mmol) in DCM(1.0 mL) is added a solution of of GC2 (0.40 g, 0.44 mmol),4-nitrophenol (0.61 g, 4.4 mmol) and TEA (0.73 mL, 5.23 mmol) in DCM (5mL) at −78° C. After stirring for 30 minutes, the mixture is warmed toroom temperature, diluted with DCM (20 mL), and washed with water (20mL×3). The combined aqueous layers are extracted with DCM (20 mL×2) andthe combined organic layers are dried over anhydrous magnesium sulfate,filtered, concentrated, and purified by silica gel column chromatography(EA/PE with 1% TEA=1/5 to 1/2) to give 82 (0.30 g, 59% yield) as a whitesolid. (MS: [M+H]⁺ 1118.9)

Step 2: CC2

To a solution of 82 (0.2 g, 0.18 mmol) in THF (1.0 mL), 4 Å MS (0.05 g)and TEA (0.12 mL, 0.89 mmol) is added AC1 (0.12 g, 0.21 mmol). Afterstirring for 12 hours, the mixture is diluted with THF (2 mL), filtered,and concentrated. The residue is then dissolved in DCM (5.0 mL) beforewater (0.1 mL) and a solution of DCA (0.46 mL) in DCM (5.0 mL) areadded. After stirring for 15 minutes, the mixture is neutralized withTEA to ˜pH 7 before concentrated and purified by reversed-phase C18silica gel column chromatography (MeCN with 0.5% TEA/water=0% to 40%) togive CC2 (0.11 g, 44% yield) as a white solid. (MS: [M+H]⁺ 1226.0)

EXAMPLES Example A: Synthesis of E5

Step 1: C4

To a solution of A4 (1.0 g, 1.01 mmol) and G1 (1.07 g, 2.02 mmol) inMeCN (10 mL) is added Py⋅DCA (420 mg, 2.02 mmol). After stirring at 20°C. for 2 hours, TBHP (70% in water, 0.65 mL, 5.05 mmol) is added and themixture is stirred for 1 hour before sodium bicarbonate aqueous solution(50 mL) is added. The mixture is extracted with ethyl acetate (100 mL)and the organic layer is washed with brine (30 mL), dried with anhydroussodium sulfate, filtered, and concentrated. The residue is thendissolved in a mixture of DCM (20 mL), TFA (1.0 mL) and triethylsilane(5.0 mL). After stirring for 2 hours, the mixture is neutralized withsolid sodium bicarbonate to ˜pH 7. The mixture is then filtered and thesolid is washed with EA (50 mL×3). The filtrate is concentrate andpurified by preparative HPLC (MeCN with 0.1% TEA/water=0% to 30%) togive C4⋅TEA as a white solid (620 mg, 49% yield). (MS: [M+H]⁺ 1131.1)

Step 2: Phosphodiester 83

To a solution of C4 (583 mg, 0.515 mmol) in Py (10 mL) is added DMOCP(583 mg, 3.16 mmol) at 20° C. After stirring for 2 hours, iodine (654mg, 2.58 mmol) is added and the mixture is stirred for 1 hour beforesaturated sodium sulfate aqueous solution (30 mL) and saturated sodiumbicarbonate aqueous solution (30 mL) is added. The mixture is thenextracted with EA (100 mL), washed with brine (60 mL), dried overanhydrous sodium sulfate, filtered, concentrated, and purified byreversed-phase silica gel column chromatography (MeCN with 0.1%TEA/water=20% to 40%) to give 83⋅TEA as a white solid (172.0 mg, 31%yield). (MS: [M+H]⁺ 1076.1)

Step 3: Bisphosphodiester 84

A solution of 83 (100 mg, 0.093 mmol) in MeOH (1.0 mL) and ammoniumhydroxide (1.0 mL) is stirred at 50° C. for 12 hours. The mixture isthen concentrated and purified by reverse-phase silica gel columnchromatography (MeCN with 0.1% TEA/water=20% to 40%) to give 84 as ayellow solid (27.0 mg, 32% yield). (MS: [M+H]⁺ 902.5)

Step 4: E5

A solution of 84 (27 mg, 0.030 mmol) in TEA⋅3HF (10 mL) is stirred at50° C. for 3 hours. The mixture is then neutralized with coldtriethylammonium bicarbonate to ˜pH 7, concentrated, and purified by aC18 reverse-phase silica gel column chromatography (MeCN with 0.1%TEA/water=0% to 20%) to give E5⋅TEA as a white solid (5.2 mg, 26%yield). (MS: [M+H]⁺ 673.7)

Example B: Synthesis of E15-E18

Step 1: C5

To a mixture of A4 (500 mg, 0.50 mmol, co-evaporated with MeCN 5 mL×1and toluene 10 mL×2) and G1 (322 mg, 0.61 mmol, co-evaporated with MeCN5 mL×1 and toluene 10 mL×2) is added tetrazole (0.45 M in MeCN, 4.0 mL).After stirring at 25° C. for 2 hours, DDTT (240 mg, 1.2 mmol) is addedand the mixture is stirred for 16 hour before filtered and concentrated.The residue is then dissolved in DCM (10 mL) followed by addition ofwater (0.1 mL) and DCA (0.21 mL). After stirring for 10 minutes, TEA (1mL) is added and the mixture is concentrate and purified byreverse-phase silica gel column chromatography (MeCN with 0.1%TEA/water=0% to 100%) to give C5⋅TEA as a white solid (250 mg, 35%yield). (MS: {[M+2H]²⁺)/2 574.6)

Step 2: Phosphorothioate 85

To a solution of C5 (600 mg, 0.423 mmol, co-evaporated with Py 3 mL×2)in Py (5.0 mL) is added DMOCP (313 mg, 1.69 mmol) at 25° C. Afterstirring for 2 hours, 3H-1,2-benzodithiol-3-one (142 mg, 0.85 mmol) isadded and the mixture is stirred for 2 hours before sodium bicarbonateaqueous solution (5%, 10 mL) is added. The mixture is then extractedwith EA (10 mL×3). The combined organic layers are dried with anhydroussodium sulfate, filtered, concentrated, and purified by reverse-phaseHPLC (MeCN with 0.1% TEA/water=0 to 100%) to give four diastereomers of85⋅TEA as white solids. Isomer 1 (28 mg) (MS: [M+H]⁺ 1160.9); Isomer 2(25 mg) (MS: [M+H]⁺ 1160.9); Isomer 3 (50 mg) (MS: [M+H]⁺ 1160.9);Isomer 4 (52 mg) (MS: [M+1]⁺ 1160.9)

Step 3: E15-E18

Each of the isomers of 85⋅TEA (25 mg, 0.022 mmol) in ammonium hydroxide(5.6 mL) and MeOH (4.0 mL) is stirred at 50° C. for 16 hours. Themixture is then purged with nitrogen at room temperature for 5 minutesbefore concentrated. The residue is dissolved in TEA (0.5 mL) and Py(0.2 mL), and TEA⋅3HF (0.7 mL) is added. After stirring at 50° C. for 24hours, triethylammonium bicarbonate aqueous solution (1M, 5 mL) is addedand the mixture is purified by a reverse-phase silica gel columnchromatography (MeCN with 0.1% TEA/water=0% to 30%) to give E15-E18 aswhite solids.

Example C: Synthesis of E24

Step 1: Phosphoramidate 44

To a solution of C3 (16 mg, 0.014 mmol, co-evaporation with Py 1 mL×3)in Py (0.5 mL) is added DMOCP (10.4 mg, 0.056 mmol). After stirring for15 minutes, NIS (4.1 mg, 0.0183 mmol) and morpholine (0.012 mL, 0.141mmol) are added and the mixture is stirred for 1 hour before sodiumbisulfite aqueous solution (0.14%, 1 mL) and sodium bicarbonate (80 mg)is added. The mixture is then extracted with DCM (5 mL×3), dried overanhydrous sodium sulfate, and concentrated. The residue is then stirredin MeCN (0.5 mL) and t-butylamine (0.5 mL) at room temperature for 15minutes before concentrated. The resulting residue is then co-evaporatedwith MeCN (1 mL×3) and purified by HPLC to give 86 as a white solid (2.4mg, 15%). (MS: [M+H]⁺ 1146.2)

Step 2: E24

To 86 (2.4 mg, 0.0021 mmol) is added methylamine (33% in EtOH, 0.3 mL).After stirring at room temperature for 16 hours, the mixture isconcentration and the residue is stirred in a mixture of TEA and TEA⋅3HFin THF (0.036 mL/0.018 mL/0.3 mL) at 35° C. for 18 hours. MeCN (1.0 mL)is then added and the solid is collected by centrifugation, washed withMeCN (1 mL×2) to give E24 as a white solid (0.6 mg, 38% yield). (MS:[M+H]⁺ 744.0)

Example D: Synthesis of E25

Step 1: Boranophosphate 87

To a solution of C3 (100 mg, 0.088 mmol, co-evaporated with Py 4 mL×3)in Py (3 mL) is added DMOCP (57 mg, 0.337 mmol). After stirring for 15minutes, BSTFA (0.10 mL, 0.371 mmol) is added dropwise and the mixtureis stirred for 20 minutes before borane N,N-diisopropylethylaminecomplex (0.092 mL, 0.530 mmol) is added. The mixture is then stirred for3 hours before concentrated and purified by silica gel columnchromatography (MeOH/DCM=1/19 to 1/9) to give semi-pure CE-protected 87as a yellow solid. The semi-pure CE-protected 87 obtained above isstirred in a mixture of MeCN (1 mL) and t-butylamine (0.5 mL) for 10minutes before concentrated. The residue is then co-evaporated with MeCN(4 mL×3) and purified by reverse-phase HPLC (MeCN with 0.1%TEA/water=40% to 90%) to give 87 as a white solid (11 mg, 12% over twosteps). (MS: [M]⁻ 1073.2)

Step 2: E25

To 87 (5.7 mg, 0.0053 mmol) is added methylamine (33% in EtOH, 1 mL).After stirring at room temperature for 18 hours, the mixture isconcentration and the residue is stirred in a mixture of TEA (0.08 mL)and TEA⋅3HF (0.04 mL) in THF (0.5 mL) at 35° C. for 18 hours. MeCN (1.2mL) is then added and the solid is collected by centrifugation, purifiedby reverse-phase HPLC (MeCN with 0.1% TFA/water=0% to 20%) to give E25as a white solid (2.5 mg, 61% yield) (MS: [M]⁻ 671.2)

Example E: Synthesis of EB1 and EB2

Step 1: CB1

To a solution of GB3 (160 mg, 0.25 mmol, co-evaporated with MeCN 1 mL×3)and pyridinium trifluoroacetate (35 mg, 0.39 mmol, co-evaporation withMeCN 1 mL×3) in MeCN (1 mL) treated with 3 Å MS (500 mg) for 30 minutesis added a solution of A4 (355 mg, 0.36 mmol, co-evaporated with MeCN 1mL×3) in MeCN (1 mL) treated with 3 Å MS (700 mg) for 30 minutes. Afterstirring at room temperature for 2 hours, TBHP (5.5 M in decane, 0.164mL, 0.9 mmol) is added and the mixture is stirred for 30 minutes beforesodium bisulfite aqueous solution (33%, 0.15 mL) is added at 0° C. Themixture is then concentrated and the residue is dissolved in DCM (4.8mL) followed by addition of water (0.054 mL) and dichloroacetic acid (6%in methylene chloride, 4.8 mL). After stirring for 10 min, the Py (1.5mL) is added and the mixture is concentrated and purified by silica gelcolumn chromatography (MeOH/DCM=1/10 to 1/4 with 1% Py) to give CB1⋅Pyas a white solid (213 mg, 66% yield).

Step 2: Phosphorothioate 88

To a solution of CB1 (60 mg, 53 μmol) in Py (1 mL) is added DMOCP (30mg, 162.5 μmol). After stirring for 10 minutes, water (0.027 mL) and3H-1,2-benzodithiol-3-one (13 mg, 0.077 mmol) are added. The mixture isstirred for 5 minutes before pouring into a solution of sodiumbicarbonate (210 mg) in water (7.5 mL). After stirring for 5 minutes,the mixture is extracted by EA/diethyl ether (1:1, 10 mL×3). Thecombined organic layers are concentrated to give a yellow solid (100mg). To a solution of the yellow solid obtained above in MeCN (0.5 mL)is added tert-butylamine (0.5 mL). After stirring for 10 minutes, themixture is concentrated and purified by HPLC (MeCN/water with 0.1% TFA:50% to 100%) to give two diastereomers of 88. Isomer 1 (7 mg) ([M+H]⁺977.0); Isomer 2 (16 mg) (MS: [M+H]⁺ 977.0)

Step 3: EB1 and EB2

To the Isomer 1 of 88 (7 mg) is added methylamine (33% in ethanol, 1mL). After stirring at room temperature for 12 hours, the mixture isconcentrated and the residue is dissolved in TFA aqueous solution (3%v/v, 1 mL). After stirring for 2 hours, the mixture is concentrated andpurified by HPLC (MeCN/water with 0.1% TFA, 0% to 45%) to give EB1 as awhite solid (2.5 mg, 57% yield). (MS: [M+H]⁺ 689.0)

To the Isomer 2 of 63 (16 mg) is added methylamine (33% in ethanol, 2mL) at 0° C. After stirring at room temperature for 12 hours, themixture is concentrated and the residue is co-evaporated with a mixtureof Py/TEA (5 mL/2 mL×3) before dissolved in Py (0.04 mL). TEA (0.25 mL)and TEA⋅3HF (0.15 mL are then added. After stirring at 55° C. for 3hours, acetone (2 mL) is added. The solid is collected (10 mg) byfiltration and purified by HPLC (MeCN/water with 0.1% TFA, 0% to 30%) togive EB2 as a white solid (5 mg, 45% yield). (MS: [M+H]⁺ 689.0, [M−H]⁻687.0)

Example F: Synthesis of EC25

To a solution of E1⋅TEA salt (10 mg, 0.0114 mmol) in water (0.3 mL) isadded 2-chloroacetaldehyde (0.015 mL, 0.118 mmol) and sodium hydroxideaqueous solution (1 M, 0.012 mL, 0.012 mmol). After stirring at 37° C.for 18 hours, the mixture is concentrated and purified by reverse-phaseHPLC (MeCN/water with 0.1% TFA=0% to 30%) to give EC26 as a white solid.(MS: [M]⁻ 697.1)

The following compounds are prepared essentially by the methods above.

TABLE 3 Examples E1 to E25, EA1 to EA11, EB1 to EB7, and EC1 to EC24Example Intermediate 1 Intermediate 2 Structure Reference of PreparationE1

Example A E2

A2

Example A E3

A2

Example A E4

Example A E5 G1

Example A E6

Example A E7 C1

Example A E8

Example A E9 C2

Example A E10

A2

Example A E11 G6 A2

Example A E12

A1

Example B E13 G2 A1

Example B E14 G2 A1

Example B E15 G1 A4

Example B E16 G1 A4

Example B E17 G1 A4

Example B E18 G1 A4

Example B E19 G1

Example B E20

Example B E21 G7 A3

Example B E22 G7 A3

Example B E23 G7 A3

Example B E24

Example C E25 C3

Example D EA1 G7 A3

Example A EA2

A1

Example A EA3 G1

Example A EA4 G1

Example B EA5 G1 AA2

Example B EA6 G7 A1

Example B EA7 G7 A1

Example B EA8 G7 A1

Example B EA9 C3

Example D EA10

Example A EA11 CA1

Example D EB1

A4

Example E EB2 GB3 A4

Example E EB3 GB3 A2

Example E EB4 GB3 A2

Example E EB5 GB3

Example E EB6

A2

Example E EB7 GB2 A2

Example E EC1

A4

Example E EC2 GC3 A4

Example E EC3 GC3 A4

Example A EC4 GB3

Example E EC5 GB3 GC4

Example E E6

A4

Example E EC7 AC3 A4

Example E EC8 AC3 GC4

Example E EC9 AC3 GC4

Example E EC10 GC3

Example A EC11 G1

Example A EC12

A2

Example A EC13 GA1

Example A EC14 G7 AC4

Example E EC15 G7 AC4

Example E EC16 GA1

Example A EC17 G1 AC2

Example E EC18 G1 AC2

Example E EC19 G1

Example A EC20 G1

Example E EC21 GC3 AC6

Example E EC22

Example A EC23

Example A EC24

A2

Example A EC25

Example F EC26

Example F

Selected physical data of the example compounds are summarized below.

TABLE 4 Physical data of cyclic dinucleotide and analogs characteristic³¹P ¹H NMR data NMR data MS data Example Structure δ (ppm)* δ (ppm)* m/zE1

8.58 (s, 1H) 8.56 (s, 1H) 8.16 (s, 1H) 6.45 (s, 1H) 6.22 (d, J = 8.5 Hz,1H) 5.89 (m, 1H) 5.31 (m, 1H) 50 °C.   0.1 −0.9 50 °C. [M + H]⁺ 675.1 E2

[M + H]⁺ 659.0 E3

8.31 (s, 1H) 8.29 (s, 1H) 7.88 (s, 1H) 6.19 (s, 1H) 5.92 (d, J = 8.7 Hz,1H) 5.71 (ddd, J = 8.4, 8.3, 4.3 Hz, 1H) 5.09 (ddd, J = 9.8, 6.9, 4.2Hz, 1H) Na⁺ salt in D₂O −1.2 −2.5 Na⁺ salt in D₂O [M − H]⁻ 687.2 E4

8.24 (s, 1H) 8.18 (s, 1H) 7.97 (s, 1H) 6.10 (d, J = 1.4 Hz, 1H) 5.97 (d,J = 8.4 Hz, 1H) 5.63 (ddd, J = 8.1, 7.9, 4.2 Hz, 1H) 5.09 (m, 1H) −1.5−2.4 [M + H]⁺ 713.2 E5

8.20 (s, 1H) 7.95 (s, 1H) 7.47 (d, J = 3.8 Hz, 1H) 6.77 (d, J = 3.8 Hz,1H) 6.08 (d, J = 4.7 Hz, 1H) 5.80 (d, J = 4.9 Hz, 1H) in D₂O—CH₃CN [M +H]⁺ 673.7 E6

8.25 (s, 1H) 8.23 (s, 1H) 8.02 (s, 1H) 6.18 (s, 1H) 5.96 (d, J = 8.6 Hz,1H) 5.43 (td, J = 8.1, 3.9 Hz, 1H) 52.4 −2.4 [M + H]⁺ 691.0 E7

8.29 (s, 1H) 8.26 (s, 1H) 7.85 (s, 1H) 6.17 (s, 1H) 5.92 (d, J = 8.5 Hz,1H) 5.61 (ddd, J = 7.9, 7.9, 4.0 Hz, 1H) 5.20 (ddd, J = 8.8, 8.8, 4.1Hz, 1H) 55.1 −2.5 [M + H]⁺ 691.0 E8

8.59 (s, 1H) 8.18 (s, 1H) 8.01 (s, 1H) 6.18 (s, 1H) 5.98 (s, 1H) 55.1−1.5 [M + H]⁺ 691.0 E9

8.35 (s, 1H) 8.13 (s, 1H) 7.95 (s, 1H) 6.12 (s, 1H) 5.92 (s, 1H) 54.4−1.6 [M + H]⁺ 691.0 E10

8.19 (s, 1H) 8.10 (s, 1H) 7.83 (s, 1H) 6.00 (d, J = 3.7 Hz, 1H) 5.84 (d,J = 5.0 Hz, 1H) 33.8 −0.4 [M + H]⁺ 657.2 E11

8.08 (s, 1H) 8.01 (s, 1H) 7.85 (s, 1H) 6.00 (1H) 5.76 (1H) 32.1 −0.3[M + H]⁺ 657.2 E12

[M + H]⁺ 707.0 E13

[M + H]⁺ 707.0 E14

[M + H]⁺ 707.0 E15

$\frac{\left\lbrack {M + {2H}} \right\rbrack^{2 +}}{2}$ 354.0 E16

8.06 (s, 1H) 8.03 (s, 1H) 7.23 (d, J = 3.7 Hz, 1H) 6.59 (d, J = 3.7 Hz,1H) 6.10 (d, J = 6.4 Hz, 1H) 5.87 (d, J = 8.5 Hz, 1H) 5.26 (ddd, J =8.5, 6.8, 4.2 Hz, 1H) 5.06 (ddd, J = 8.0, 4.7, 2.8 Hz, 1H) in DMSO-d₆57.9 51.7 in DMSO-d₆ [M + H]⁺ 706.1 E17

[M + H]⁺ 706.1 E18

8.23 (s, 1H) 8.19 (s, 1H) 7.43 (d, J = 3.8 Hz, 1H) 6.52 (d, J = 3.8 Hz,1H) 6.19 (s, 1H) 5.89 (d, J = 8.5 Hz, 1H) 5.23 (m, 1H) 4.95 (m, 1H) inCD₃CN 51.3 49.1 in CD₃CN [M + H]⁺ 706.1 E19

8.84 (s, 1H) 8.58 (s, 1H) 6.58 (d, J = 4.6 Hz, 1H) 6.24 (d, J= 8.5 Hz,1H) 5.79 (m, 1H) 5.60 (m, 1H) in D₂O 59.9 56.7 in D₂O [M + H]⁺ 708.2 E20

8.57 (s, 1H) 7.83 (d, J = 3.8 Hz, 1H) 7.62 (d, J = 3.8 Hz, 1H) 7.06 (d,J = 3.8 Hz, 1H) 6.84 (d, J = 3.8 Hz, 1H) 6.61 (d, J = 6.2 Hz, 1H) 6.48(d, J = 8.4 Hz, 1H) 5.69 (ddd, J = 8.0, 4.6, 3.1 Hz, 1H) 5.63 (ddd, J =12.1, 8.4, 4.3 Hz, 1H) in D₂O 59.3 57.8 in D₂O [M + H]⁺ 705.3 E21

8.34 (s, 1H) 7.62 (d, J = 3.8 Hz, 1H) 7.33 (d, J = 3.8 Hz, 1H) 6.94 (d,J = 3.8 Hz, 1H) 6.28 (d, J = 3.8 Hz, 1H) 6.13 (d, J = 5.4 Hz, 1H) 6.10(d, J = 8.4 Hz, 1H) in DMSO-d₆ 54.9 50.1 in DMSO-d₆ [M + H]⁺ 705.1 E22

8.35 (s, 1H) 7.65 (d, J = 3.8 Hz, 1H) 7.09 (d, J = 3.8 Hz, 1H) 6.97 (d,J = 3.7 Hz, 1H) 6.28 (d, J = 3.7 Hz, 1H) 6.11 (d, J = 7.9 Hz, 1H) 6.07(d, J = 8.5 Hz, 1H) 5.28 (dd, J = 8.6, 4.4 Hz, 1H) 5.01 (m, 1H) inDMSO-d₆ 59.7 58.9 in DMSO-d₆ [M + H]⁺ 705.1 E23

8.35 (s, 1H) 7.69 (d, J = 3.8 Hz, 1H) 7.07 (d, J = 3.8 Hz, 1H) 6.95 (d,J = 3.7 Hz, 1H) 6.31 (d, J = 3.7 Hz, 1H) 6.13 (d, J = 7.2 Hz, 1H) 6.06(d, J = 8.5 Hz, 1H) 5.05 (m, 1H) 4.99 (m, 1H) in DMSO-d₆ 58.5 50.9 inDMSO-d₆ [M + H]⁺ 705.1 E24

8.32 (s, 1H) 8.02 (s, 1H) 7.98 (s, 1H) 6.18 (s, 1H) 6.07 (d, J = 8.7 Hz,1H) 5.63 (ddd, J = 9.1, 5.0, 5.0, 1H)   5.6 −1.4 [M + H]⁺ 744.0 E25

8.55 (s, 1H) 8.45 (s, 1H) 8.29 (s, 1H) 6.27 (s, 1H) 5.96 (d, J = 8.3 Hz,1H) 5.56 (m, 1H) 5.03 (ddd, J = 9.4, 6.5, 4.2 Hz, 1H) −1.3 [M]⁻ 671.2EA1

8.07 (s, 1H) 7.37 (d, J = 3.8 Hz, 1H) 7.09 (d, J = 3.8 Hz, 1H) 6.65 (d,J = 3.7 Hz, 1H) 6.35 (d, J = 3.7 Hz, 1H) 6.08 (d, J = 7.4 Hz, 1H) 6.04(d, J = 8.4 Hz, 1H) 4.85 (m, 1H) 4.81 (m, 1H) in DMSO-d₆ 0.98 0.96 inDMSO-d₆ [M + H]⁺ 673.1 EA2

8.29 (s, 1H) 8.25 (s, 1H) 7.89 (s, 1H) 6.17 (s, 1H) 6.01 (d, J = 8.6 Hz,1H) 5.69 (ddd, J = 8.7, 8.7, 4.0 Hz, 1H) 5.09 (m, 1H) Na⁺ salt in D20−1.1 −2.3 Na⁺ salt in D₂O [M + H]⁺ 757.0 [M − H]⁻ 755.0 EA3

8.26 (s, 1H) 8.23 (s, 1H) 7.85 (s, 1H) 6.27 (s, 1H) 5.92 (d, J = 8.6 Hz,1H) 5.63 (m, 1H) 5.10 (m, 1H) −1.6 −2.3 [M + H]⁺ 713.0 [M − H]⁻ 711.0EA4

8.19 (s, 1H) 8.18 (s, 1H) 8.15 (brs, 1H) 6.25 (d, J = 3.3 Hz, 1H) 5.92(d, J = 8.5 Hz, 1H) 5.24 (m, 2H) in CD₃CN 55.4 53.5 in CD₃CN [M + H]⁺707.1 EA5

59.6 58.1 in CD₃CN [M + H]⁺ 707.1 EA6

8.62 (s, 1H) 8.55 (s, 1H) 7.69 (d, J = 3.7 Hz, 1H) 6.59 (d, J = 3.7 Hz,1H) 6.41 (d, J = 8.4 Hz, 1H) 6.37 (d, J = 4.6 Hz, 1H) 5.59 (m, 1H) 5.51(m, 1H) in D₂O 58.1 56.0 in D₂O [M + H]⁺ 706.0 EA7

56.7 55.6 in CD₃CN [M + H]⁺ 706.0 EA8

53.4 48.7 in CD₃CN [M + H]⁺ 705.9 EA9

8.41 (s, 1H) 8.26 (s, 1H) 7.85 (s, 1H) 6.17 (s, 1H) 5.90 (d, J = 8.6 Hz,1H) 5.76 (ddd, J = 8.8, 8.6, 4.1 Hz, 1H) 5.08 (m, 1H) [M]⁻ 671.2 EA10

8.32 (s, 1H) 8.26 (s, 1H) 7.86 (s, 1H) 6.16 (s, 1H) 5.92 (d, J = 8.5 Hz,1H) 5.67 (m, 1H) 5.21 (m, 1H) [M]⁻ 671.2 EA11

8.43 (s, 1H) 8.41 (s, 1H) 6.19 (s, 1H) 6.07 (d, J = 8.2 Hz, 1H) [M + H]⁻669.2 EB1

8.16 (s, 1H) 7.48 (d, J = 3.3 Hz, 1H) 7.18 (d, J = 3.3 Hz, 1H) 6.50 (d,J = 3.5 Hz, 1H) 6.49 (d, J = 3.5 Hz, 1H) 6.29 (d, J = 5.1 Hz, 1H) 6.09(d, J = 8.7 Hz, 1H) 5.49 (m, 1H) 5.17 (m, 1H) 57.7 −0.2 [M + H]⁺ 689.0EB2

8.16 (s, 1H) 7.33 (d, J = 3.8 Hz, 1H) 7.07 (d, J = 3.8 Hz, 1H) 6.44 (d,J = 3.8 Hz, 1H) 6.33 (d, J = 3.8 Hz, 1H) 6.27 (d, J = 2.5 Hz, 1H) 6.01(d, J = 8.5 Hz, 1H) 5.48 (dd, J = 8.6, 8.5, 4.1 Hz, 1H) 5.00 (ddd, J =7.1, 7.1, 4.7 Hz, 1H) 52.0 −1.1 [M + H]⁺ 689.0 EB3

8.26 (s, 1H) 8.25 (s, 1H) 6.95 (d, J = 3.8 Hz, 1H) 6.29 (d, J = 3.8 Hz,1H) 6.15 (s, 1H) 5.93 (d, J = 8.7 Hz, 1H) 5.55 (m, 1H) 4.99 (m, 1H) 51.5−1.3 [M + H]⁺ 690.0 EB4

8.46 (s, 1H) 8.26 (s, 1H) 7.03 (d, J = 3.7 Hz, 1H) 6.38 (d, J = 3.7 Hz,1H) 6.17 (d, J = 2.7 Hz, 1H) 5.99 (d, J = 8.4 Hz, 1H) 5.63 (m, 1H) 5.11(ddd, J = 6.7, 6.6, 4.5 Hz, 1H) 56.1 −0.7 [M + H]⁺ 690.0 EB5

8.26 (s, 1H) 8.23 (s, 1H) 6.94 (d, J = 3.7 Hz, 1H) 6.26 (d, J = 3.7 Hz,1H) 6.14 (s, 1H) 5.92 (d, J = 8.5 Hz, 1H) 5.55 (m, 1H) 5.00 (m, 1H) 51.5−1.3 [M + H]⁺ 734.0 [M − H]⁻ 732.0 EB6

8.55 (s, 1H) 8.26 (s, 1H) 7.55 (s, 1H) 6.22 (s, 1H) 6.08 (d, J = 8.6 Hz,1H) 5.81 (ddd, J = 10.4, 8.6, 4.0 Hz, 1H) 5.32 (ddd, J = 8.9, 8.8, 4.3Hz, 1H) 54.1 52.5 [M − H]⁻ 705.0 EB7

8.59 (s, 1H) 8.26 (s, 1H) 7.79 (s, 1H) 6.21 (s, 1H) 6.05 (d, J = 8.6 Hz,1H) 5.88 (ddd, J = 10.5, 8.6, 4.1 Hz, 1H) 5.31 (ddd, J = 8.4, 8.4, 4.1Hz, 1H) 55.4 54.2 [M − H]⁻ 705.0 EC1

8.15 (s, 1H) 7.89 (s, 1H) 7.35 (d, J = 3.8 Hz, 1H) 6.27 (s, 1H) 6.26 (d,J = 3.8 Hz, 1H) 5.98 (d, J = 8.6 Hz, 1H) 5.75 (ddd, J = 9.2, 9.1, 4.1Hz, 1H) 5.07 (m, 1H) 52.0 −1.3 [M + H]⁺ 772.1 EC2

8.15 (s, 1H) 7.97 (s, 1H) 7.56 (d, J = 3.8 Hz, 1H) 6.37 (d, J = 3.8 Hz,1H) 6.30 (d, J = 3.1 Hz, 1H) 6.02 (d, J = 8.5 Hz, 1H) 5.78 (ddd, J =12.6, 8.5, 4.1 Hz, 1H) 5.16 (m, 1H) 56.3 −0.6 [M + H]⁺ 772.1 EC3

8.15 (s, 1H) 7.87 (s, 1H) 7.33 (d, J = 3.8 Hz, 1H) 6.27 (s, 1H) 6.24 (d,J = 3.8 Hz, 1H) 5.98 (d, J = 8.6 Hz, 1H) 5.68 (ddd, J = 8.5, 8.45, 4.0Hz, 1H) 5.08 (ddd, J = 7.7, 7.5, 4.5 Hz, 1H) −1.4 −2.4 [M + H]⁺ 756.2EC4

7.03 (d, J = 3.7 Hz, 1H) 6.88 (m, 1H) 6.34 (d, J = 3.7 Hz, 1H) 6.31 (d,J = 3.7 Hz, 1H) 6.08 (m, 1H) 5.93 (m, 1H) 5.05 (m, 1H) 4.98 (m, 1H) inDMSO-d₆ /D₂O 61.1 −0.4 [M + H]⁺ 705.0 EC5

7.02 (d, J = 3.7 Hz, 1H) 6.92 (d, J = 3.7 Hz, 1H) 6.33 (m, 2H) 6.09 (d,J = 8.7 Hz, 1H) 5.92 (d, J = 7.9 Hz, 1H) 4.94 (m, 1H) 4.81 (m, 1H) 4.60(m, 1H) 4.43 (m, 1H) in DMSO-d₆ /D₂O 53.1 −0.8 [M + H]⁺ 704.9 EC6

8.11 (s, 1H) 8.11 (s, 1H) 7.59 (d, J = 3.8 Hz, 1H) 7.20 (d, J = 3.9 Hz,1H) 6.40 (m, 2H) 6.22 (m, 2H) 5.23 (ddd, J = 9.0, 8.8, 4.2 Hz, 1H) 4.94(m, 1H) 52.9 −1.0 [M + H]⁺ 673.0 EC7

8.103 (s, 1H) 8.102 (s, 1H) 7.67 (d, J = 3.9 Hz, 1H) 7.34 (d, J = 3.9Hz, 1H) 6.58 (d, J = 3.8 Hz, 1H) 6.51 (d, J = 3.8 Hz, 1H) 6.42 (d, J =8.4 Hz, 1H) 6.23 (d, J = 6.5 Hz, 1H) 59.1  0.1 [M + H]⁺ 673.0 EC8

8.27 (s, 2H) 8.05 (s, 1H) 7.47 (d, J = 3.8 Hz, 1H) 6.92 (d, J = 3.7 Hz,1H) 6.65 (d, J = 3.7 Hz, 1H) 6.30 (m, 2H) 5.99 (d, J = 8.2 Hz, 1H) 5.01(m, 1H) 4.85 (m, 1H) in DMSO-d₆ 49.5  0.9 in DMSO-d₆ [M + H]⁺ 689.2 EC9

8.29 (s, 2H) 8.04 (s, 1H) 7.50 (d, J = 3.8 Hz, 1H) 6.82 (d, J = 3.7 Hz,1H) 6.59 (d, J = 3.8 Hz, 1H) 6.35 (d, J = 3.7 Hz, 1H) 6.29 (d, J = 8.5Hz, 1H) 5.98 (d, J = .5 Hz, 1H) 5.11 (m, 1H) 4.40 (m, 1H) in DMSO-d₆57.1  1.4 in DMSO-d₆ [M + H]⁺ 689.1 EC10

8.28 (s, 1H) 8.26 (s, 1H) 7.83 (s, 1H) 6.26 (s, 1H) 5.96 (d, J = 8.6 Hz,1H) 5.76 (ddd, J = 8.6, 8.6, 4.1 Hz, 1H) 5.09 (m,1H) −1.6 −2.7 [M + H]⁺771.2 EC11

8.32 (s, 1H) 8.27 (s, 1H) 7.93 (s, 1H) 6.47 (m, 1H) 5.97 (d, J = 8.3 Hz,1H) 5.58 (m, 1H) 5.19 (m, 1H) [M + H]⁺ 659.0 [M − H]⁻ 656.8 EC12

8.30 (s, 1H) 8.25 (s, 1H) 7.84 (s, 1H) 6.16 (s, 1H) 5.88 (d, J = 8.4 Hz,1H) 5.73 (ddd, J = 8.0, 8.0, 8.0 Hz, 1H) 5.08 (m, 1H) −1.0 −2.4 [M + H]⁺703.0 EC13

8.36 (s, 1H) 8.18 (s, 1H) 7.84 (s, 1H) 6.29 (d, J = 8.1 Hz, 1H) 5.99 (d,J = 8.4 Hz, 1H) 5.38 (ddd, J = 8.1, 7.9, 4.4 Hz, 1H) 5.09 (ddd, J = 8.3,4.2, 4.2 Hz, 1H) −1.3 −1.8 [M + H]⁺ 757.0 EC14

8.69 (s, 1H) 8.13 (s, 1H) 6.88 (d, J = 3.8 Hz, 1H) 6.34 (d, J = 3.8 Hz,1H) 6.28 (d, J = 8.1 Hz, 1H) 6.10 (d, J = 8.1 Hz, 1H) 5.17 (m, 2H) 56.7−1.5 [M + H]⁺ 689.9 EC15

8.35 (s, 1H) 7.93 (s, 1H) 6.77 (d, J = 3.8 Hz, 1H) 6.25 (d, J = 8.1 Hz,1H) 6.17 (d, J = 3.8 Hz, 1H) 6.10 (d, J = 8.1 Hz, 1H) 5.19 (ddd, J =8.6, 4.6, 4.6 Hz, 1H) 5.14 (ddd, J = 8.5, 4.4, 4.4 Hz, 1H) 52.1 −1.6[M + H]⁺ 690.0 EC16

8.03 (s, 1H) 7.68 (m, 1H) 7.58 (d, J = 8.0 Hz, 1H) 7.44 (d, J = 3.4 Hz,1H) 7.20 (m, 2H) 6.59 (t, J = 6.5 Hz, 1H) 6.55 (d, J = 3.4 Hz, 1H) 6.05(d, J = 8.5 Hz, 1H) 5.55 (m, 1H) 5.20 (m, 1H) −1.0 −1.1 [M + H]⁺ 723.1EC17

8.03 (s, 1H) 7.67 (m, 1H) 7.58 (m, 1H) 7.44 (d, J = 3.4 Hz, 1H) 7.18 (m,2H) 6.57 (t, J = 6.4 Hz, 1H) 6.52 (m, 1H) 6.02 (d, J = 8.5 Hz, 1H) 5.57(m, 1H) 5.20 (m, 1H) 53.0 −1.0 [M + H]⁺ 657.0 [M − H]⁻ 655.0 EC18

8.16 (s, 1H) 7.68 (d, J = 7.8 Hz, 1H) 7.62 (d, J = 8.1 Hz, 1H) 7.52 (d,J = 3.5 Hz, 1H) 7.26 (m, 1H) 7.19 (m, 1H) 6.61 (m, 2H) 6.05 (d, J = 8.3Hz, 1H) 5.50 (m, 1H) 5.31 (m, 1H) 59.5 −1.0 [M + H]⁺ 657.0 [M − H]⁻655.0 EC19

8.06 (m, 1H) 7.99 (m, 1H) 7.97 (s, 1H) 7.84 (d, J = 8.3 Hz, 1H) 7.69 (d,J = 7.2 Hz, 1H) 7.59 (m, 2H) 7.32 (t, J = 7.7 Hz, 1H) 6.02 (m, 2H) 5.52(ddd, J = 8.7, 8.7, 4.2 Hz, 1H) 5.08 (m, 1H) −1.0 −1.2 [M + H]⁺ 734.0EC20

9.12 (s, 1H) 8.29 (d, J = 9.3 Hz, 1H) 8.12 (m, 5H) 8.00 (s, 2H) 7.93 (t,J = 7.6 Hz, 1H) 6.18 (m, 2H) 5.36 (m, 2H) 62.8 −0.7 [M + H]⁺ 742.1 EC21

9.09 (s, 1H) 8.28 (d, J = 9.3 Hz, 1H) 8.11 (m, 5H) 7.98 (s, 2H) 7.93 (t,J = 7.6 Hz, 1H) 6.17 (dd, J = 10.5, 5.3 Hz, 1H) 6.13 (d, J = 8.0 Hz, 1H)5.46 (m, 1H) 5.13 (m, 1H) 56.5 −0.8 [M + H]⁺ 742.0 EC22

8.21 (s, 1H) 8.14 (s, 1H) 7.82 (s, 1H) 6.19 (d, J = 7.7 Hz, 1H) 6.00 (d,J = 3.1 Hz, 1H) −0.1 [M + H]⁺ 689.0 EC23

8.15 (s, 1H) 7.91 (s, 1H) 7.44 (s, 1H) 6.17 (s, 1H) 5.88 (d, J = 9.0 Hz,1H) 5.13 (m, 2H) −1.24 [M + H]⁺ 672.9 EC24

8.31 (s, 1H) 8.26 (s, 1H) 7.84 (s, 1H) 6.17 (s, 1H) 5.86 (d, J = 8.4 Hz,1H) 5.68 (m, 1H) 5.08 (m, 1H) −1.0 −2.2 [M + H]⁺ 701.2 [M − H]⁻ 699.0EC25

9.08 (s, 1H) 8.38 (s, 1H) 7.96 (s, 1H) 7.90 (s, 1H) 7.57 (s, 1H) 6.28(s, 1H) 5.95 (d, J = 8.5 Hz, 1H) 5.64 (ddd, J = 8.3, 8.3, 4.2 Hz, 1H)5.08 (ddd, J = 8.8, 6.5, 4.3 Hz, 1H) −1.3 −2.0 [M − H]⁻ 697.1 EC26

9.15 (s, 1H) 8.40 (s, 1H) 8.02 (s, 1H) 7.90 (s, 1H) 7.63 (s, 1H) 6.32(s, 1H) 5.98 (d, J = 8.6 Hz, 1H) 5.72 (ddd, J = 8.7, 8.7, 4.1 Hz, 1H)5.11 (ddd, J = 8.6, 6.8, 4.3 Hz, 1H) −1.3 −2.3 [M − H]⁻ 779.0 *InNaH₂PO₄/Na₂HPO₄/D₂O unless otherwise mentioned.

Stereochemical information of represented compounds is given below.

TABLE 5 P-Configuration of Examples E23 and EB3 E23 [Rp, Rp]

EB3 [Rp]

Biological Testing

Serial dilutions of cGAMP analog compounds in phosphate buffer saline(PBS) are mixed with THP1 luciferase reporter cells in a 96-well plateat 0.2×10⁶/well, in the presence or absence of 1 nM of Perfringolysin O(PFO), which can facilitate compound uptake by forming open channels onthe plasma membrane. 16 hours later, 15 μL of the media from each wellis transferred to a new plate, and luminescence is measured. Foldincrease in luminescence compared to PBS stimulated cells is plottedagainst logarithm of concentrations of each compound, and EC₅₀ iscalculated using Graphpad.

TABLE 6 Activity of cyclic dinucleotides and analogs THP1-ISG-LucTHP-ISG-Luc with PFO, without PFO, Example EC₅₀, nM EC₅₀, μM E1 A A E2 AA E3 A A E4 A A E5 C C E6 A A E7 A A E8 B C E9 A A E10 C C E11 B C E12 AA E13 B C E14 B C E15 C C E16 A A E17 C C E18 A A E19 C C E20 B A E21 BA E22 B A E23 A A E24 B A E25 A A EA1 B C EA2 A A EA3 C C EA4 B A EA5 CC EA6 C C EA7 B A EA8 B A EA9 A A EA10 A A EA11 A A EB1 C B EB2 A A EB3A A EB4 C B EB5 B C EB6 C C EB7 C C EC1 A A EC2 B B EC3 B A EC4 C C EC5B B EC6 C C EC7 C C EC8 C C EC9 C C EC10 C C EC11 A C EC12 A A EC13 B AEC14 C C EC15 B A EC16 C C EC17 C C EC18 C C EC19 C C EC20 C C EC21 C CEC22 C C EC23 C C EC24 A A EC26 B C Activity Code A ≤100 nM A ≤30 μM B100-1000 nm B 30-100 μM C >1000 nM C >100 μM

2′3′-cGAMP can be degraded by the enzyme ecto-nucleotidepyrophosphatase/phosphodiesterase (ENPP1) which is present in fetalbovine serum (FBS) (Li et al., 2015, Nat Chem Biol, 11, 235). To test ifcGAMP analogues have improved stability, 5 μL of of synthetic cGAMPanalogues (100 μM stock) were incubated with 45 μL of FBS in a finalvolume of 50 μL at 37° C. for 1, 2 and 4 hours. At the indicated time,10 μL of the reaction mixture was taken out and mixed with 10 μLphosphate buffered saline (PBS), then heated at 95° C. to denatureproteins, which were removed by centrifugation at 13000 g for 5 minutes.The supernatants were delivered to THP1-ISG-luciferase cell line in thepresence of PFO to measure the activity of remaining cGAMP analogues, asdescribed above. Category A indicates less than 10% decrease of activityafter 4-hour incubation, B indicates 10-75% decrease of activity after4-hour incubation, and C indicates more than 75% loss of activity after4-hour incubation.

TABLE 7 Stability of cyclic dinucleotides and analogues in fetal bovineserum Reduction of activity Example after FBS incubation E1 C E2 C E3 AE6 C E7 C E8 A E9 A E16 B E18 B E20 A E21 B E22 A E23 B E25 A EA2 A EA4A EA6 A EA7 A EA8 A EA10 C EA11 A EB1 A EB2 B EB3 A EB4 A EB5 A EC1 AEC2 A EC3 B EC5 A EC13 A EC15 A EC24 A EC26 A Activity Code A <10%B >10% C >75%

The following series of prophetic examples are also compounds of thepresent invention:

TABLE 8 Structures of P1 to P15

P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

P11

P12

P13

P14

P15

The following Example compounds in Table 9 are also compoundscontemplated by the present disclosure.

TABLE 9 Additional Compounds Compound Structure PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA10

PA11

PA12

PB1

PB2

PB3

PB4

PB5

PB6

PB7

PB8

PB9

PB10

PB11

PB12

PB13

PB14

PC1

PC2

PC3

PC4

PC5

PC6

PC7

PC8

PC9

PC10

PC11

PC12

PD1

PD2

PD3

PD4

PD5

PD6

The invention claimed is:
 1. A compound, wherein the compound is ofFormula Ic:

wherein Z¹² and Z¹⁵ are N; Z¹³, Z¹⁴, Z¹⁶ and Z¹⁷ are independently CH orN; R³ is C₂alkyl functionalized with one or more halogen, thiol,hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups; R⁴ is hydroxyl; R⁹and R¹⁰ are independently hydroxyl; thiol; C₁₋₆alkyl; C₁₋₆alkylfunctionalized with one or more halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino,di(C₁₋₆alkyl)amino, or azido groups; C₁₋₆alkoxy; C₁₋₆alkoxyfunctionalized with one or more halogen, thiol, hydroxyl, carboxyl,C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azidogroups; C₃₋₅alkenyl-O—; C₃₋₅alkynyl-O—; oligo(ethylene glycol);poly(ethylene glycol); borano (—BH₃ ⁻); or —NR⁷R⁸; R⁷ and R⁸ areindependently hydrogen; C₁₋₆alkyl; C₁₋₆alkyl functionalized with one ormore halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups; cyclic—(C₁₋₆alkyl)-; cyclic —(C₁₋₆alkyl)- functionalized with one or morehalogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,amino, C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; cyclic—(C₁₋₆oxaalkyl)-; or cyclic —(C₁₋₆oxaalkyl)-functionalized with one ormore halogen, thiol, hydroxyl, carboxyl, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1, wherein Z¹³ and Z¹⁶are CH.
 3. The compound of claim 1, wherein Z¹⁴ and Z¹⁷ are N.
 4. Thecompound of claim 1, wherein Z¹³ and Z¹⁶ are CH, and Z¹⁴ and Z¹⁷ are N.5. The compound of claim 1, wherein R⁹ and R¹⁰ are independentlyhydroxyl or thiol.
 6. The compound of claim 1, wherein R⁹ and R¹⁰ arehydroxyl.
 7. The compound of claim 1, wherein R⁹ and R¹⁰ are thiol. 8.The compound of claim 1, wherein R³ is C₂alkyl functionalized with oneor more thiol, hydroxyl, carboxyl, C₁₋₆hydroxyalkoxy, amino, orC₁₋₆alkylamino groups.
 9. The compound of claim 1, wherein R³ is C₂alkylfunctionalized with one or more halogen, thiol, or hydroxyl.
 10. Acompound, wherein the compound is of Formula Ic:

wherein Z¹² and Z¹⁵ are N; Z¹³ and Z¹⁶ are CH; Z¹⁴ and Z¹⁷ areindependently CH or N; R³ is C₂alkyl functionalized with one or morehalogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups; R⁴ ishydroxyl; R⁹ and R¹⁰ are independently hydroxyl or thiol; or apharmaceutically acceptable salt thereof.
 11. The compound of claim 10,wherein R³ is C₂alkyl functionalized with one or more thiol, hydroxyl,carboxyl, C₁₋₆hydroxyalkoxy, amino, or C₁₋₆alkylamino groups.
 12. Thecompound of claim 10, wherein R³ is C₂alkyl functionalized withhydroxyl.
 13. The compound of claim 10, wherein R³ is C₂alkylfunctionalized with thiol.
 14. The compound of claim 10, wherein R³ isC₂alkyl functionalized with halogen.
 15. A compound, wherein thecompound is of Formula Ic:

wherein Z¹² and Z¹⁵ are N; Z¹³, Z¹⁴, Z¹⁶ and Z¹⁷ are independently CH orN; R³ is C₁₋₆alkyl functionalized with one or more halogen, thiol, orhydroxyl; R⁴ is hydroxyl; R⁹ and R¹⁰ are independently hydroxyl; thiol;C₁₋₆alkyl; C₁₋₆alkyl functionalized with one or more halogen, thiol,hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups; C₁₋₆alkoxy;C₁₋₆alkoxy functionalized with one or more halogen, thiol, hydroxyl,carboxyl, C₁₋₆hydroxyalkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,or azido groups; C₃₋₅alkenyl-O—; C₃₋₅alkynyl-O—; oligo(ethylene glycol);poly(ethylene glycol); borano (—BH₃ ⁻); or —NR⁷R⁸; R⁷ and R⁸ areindependently hydrogen; C₁₋₆alkyl; C₁₋₆alkyl functionalized with one ormore halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, or azido groups; cyclic—(C₁₋₆alkyl)-; cyclic —(C₁₋₆alkyl)- functionalized with one or morehalogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,amino, C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; cyclic—(C₁₋₆oxaalkyl)-; or cyclic —(C₁₋₆oxaalkyl)-functionalized with one ormore halogen, thiol, hydroxyl, carboxyl, C₁₋₆hydroxyalkoxy, amino,C₁₋₆alkylamino, or di(C₁₋₆alkyl)amino groups; or a pharmaceuticallyacceptable salt thereof.
 16. The compound of claim 15 having thestructure:

or a pharmaceutically acceptable salt thereof.
 17. The compound of claim15 having the structure:

or a pharmaceutically acceptable salt thereof.
 18. The compound of claim15 having the structure:

or a pharmaceutically acceptable salt thereof.
 19. The compound of claim15 having the structure:

or a pharmaceutically acceptable salt thereof.
 20. A compound, whereinthe compound is of Formula Ic:

wherein Z¹² and Z¹⁵ are N; Z¹³ and Z¹⁶ are CH; Z¹⁴ and Z¹⁷ areindependently CH or N; R³ is C₁₋₆alkyl functionalized with one or morehalogen, thiol, or hydroxyl; R⁴ is hydroxyl; R⁹ and R¹⁰ areindependently hydroxyl or thiol; or a pharmaceutically acceptable saltthereof.
 21. The compound of claim 20, wherein R³ is C₁₋₆alkylfunctionalized with hydroxyl.
 22. The compound of claim 20, wherein R³is C₁₋₆alkyl functionalized with thiol.
 23. The compound of claim 20,wherein R³ is C₁₋₆alkyl functionalized with halogen.
 24. Apharmaceutical composition that provides a therapeutically effectiveamount of a compound of claim 1 and one or more pharmaceuticallyacceptable excipients.
 25. A pharmaceutical composition that provides atherapeutically effective amount of a compound of claim 1 in combinationwith at least one further therapeutic agent and one or morepharmaceutically acceptable excipients.
 26. An immunogenic compositioncomprising an antigen or antigen composition and a compound of claim 1.27. A pharmaceutical composition that provides a therapeuticallyeffective amount of a compound of claim 8 and one or morepharmaceutically acceptable excipients.
 28. A pharmaceutical compositionthat provides a therapeutically effective amount of a compound of claim10 and one or more pharmaceutically acceptable excipients.
 29. Apharmaceutical composition that provides a therapeutically effectiveamount of a compound of claim 11 and one or more pharmaceuticallyacceptable excipients.
 30. A pharmaceutical composition that provides atherapeutically effective amount of a compound of claim 15 and one ormore pharmaceutically acceptable excipients.
 31. A pharmaceuticalcomposition that provides a therapeutically effective amount of acompound of claim 16 and one or more pharmaceutically acceptableexcipients.
 32. A pharmaceutical composition that provides atherapeutically effective amount of a compound of claim 17 and one ormore pharmaceutically acceptable excipients.
 33. A pharmaceuticalcomposition that provides a therapeutically effective amount of acompound of claim 18 and one or more pharmaceutically acceptableexcipients.
 34. A pharmaceutical composition that provides atherapeutically effective amount of a compound of claim 19 and one ormore pharmaceutically acceptable excipients.